Novel antibodies inhibiting c-met dimerization,  and uses thereof

ABSTRACT

The present inventions relates to a process for the selection of anti c-Met antibodies capable to inhibit both ligand-dependent and ligand-independent activation of c-Met. More particularly, said process is based on the inhibition of the c-Met dimerization. In another aspect, the present invention concerns such antibodies and compositions comprising such antibodies for the preparation of a medicament to treat cancer. Diagnosis process and kits are also part of the invention.

CROSS-REFERENCE TO PRIORITY APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/440,571, filed Mar. 9, 2009, now issued as U.S. Pat. No. 8,329,173,which claims priority under 35 U.S.C. §119 of EP 07301231.2, filed Jul.12, 2007, U.S. Provisional Application No. 60/929,789, filed Jul. 12,2007, U.S. Provisional Application No. 61/020,639, filed Jan. 11, 2008,and is a national phase of PCT/EP2008/059026, filed Jul. 10, 2008, anddesignating the United States (published in the English language on Jan.15, 2009, as WO 2009/007427 A2; the title and abstract were alsopublished in English), each hereby expressly incorporated by referencein its entirety and each assigned to the assignee hereof.

TECHNICAL FIELD

The present invention relates to novel antibodies capable of bindingspecifically to the human c-Met receptor and/or capable of specificallyinhibiting the tyrosine kinase activity of said receptor, especiallymonoclonal antibodies of murine, chimeric and humanized origin, as wellas the amino acid and nucleic acid sequences coding for theseantibodies. More particularly, antibodies according to the invention arecapable of inhibiting the c-Met dimerization. The invention likewisecomprises the use of these antibodies as a medicament for theprophylactic and/or therapeutic treatment of cancers or any pathologyconnected with the overexpression of said receptor as well as inprocesses or kits for diagnosis of illnesses connected with theoverexpression of c-Met. The invention finally comprises products and/orcompositions comprising such antibodies in combination with otherantibodies and/or chemical compounds directed against other growthfactors involved in tumor progression or metastasis and/or compoundsand/or anti-cancer agents or agents conjugated with toxins and their usefor the prevention and/or the treatment of certain cancers.

BACKGROUND OF THE INVENTION

Receptor tyrosine kinase (RTK) targeted agents such as trastuzumab,cetuximab, bevacizumab, imatinib and gefitinib inhibitors haveillustrated the interest of targeting this protein class for treatmentof selected cancers.

c-Met, is the prototypic member of a sub-family of RTKs which alsoincludes RON and SEA. The c-Met RTK family is structurally differentfrom other RTK families and is the only known high-affinity receptor forhepatocyte growth factor (HGF), also called scater factor (SF) [D. P.Bottaro et al., Science 1991, 251: 802-804; L. Naldini et al., Eur. Mol.Biol. Org. J. 1991, 10:2867-2878]. c-Met and HGF are widely expressed ina variety of tissue and their expression is normally restricted to cellsof epithelial and mesenchymal origin respectively [M. F. Di Renzo etal., Oncogene 1991, 6:1997-2003; E. Sonnenberg et al., J. Cell. Biol.1993, 123:223-235]. They are both required for normal mammaliandevelopment and have been shown to be particularly important in cellmigration, morphogenic differentiation, and organization of thethree-dimensional tubular structures as well as growth and angiogenesis[F. Baldt et al., Nature 1995, 376:768-771; C. Schmidt et al., Nature.1995:373:699-702; Tsarfaty et al., Science 1994, 263:98-101]. While thecontrolled regulation of c-Met and HGF have been shown to be importantin mammalian development, tissue maintenance and repair [Nagayama T,Nagayama M, Kohara S, Kamiguchi H, Shibuya M, Katoh Y, Itoh J, ShinoharaY., Brain Res. 2004, 5; 999(2):155-66; Tahara Y, Ido A, Yamamoto S,Miyata Y, Uto H, Hori T, Hayashi K, Tsubouchi H., J Pharmacol Exp Ther.2003, 307(1):146-51], their dysregulation is implicated in theprogression of cancers.

Aberrant signalling driven by inappropriate activation of c-Met is oneof the most frequent alteration observed in human cancers and plays acrucial role in tumorigenesis and metastasis [Birchmeier et al., Nat.Rev. Mol. Cell. Biol. 2003, 4:915-925; L. Trusolino and Comoglio P. M.,Nat. Rev. Cancer. 2002, 2(4):289-300].

Inappropriate c-Met activation can arise by ligand-dependent andindependent mechanisms, which include overexpression of c-Met, and/orparacrine or autocrine activation, or through gain in function mutation[J. G. Christensen, Burrows J. and Salgia R., Cancer Latters. 2005,226:1-26]. However an oligomerization of c-Met receptor, in presence orin absence of the ligand, is required to regulate the binding affinityand binding kinetics of the kinase toward ATP and tyrosine-containingpeptide substrates [Hays J L, Watowich S J, Biochemistry, 2004 August17, 43:10570-8]. Activated c-Met recruits signalling effectors to itsmultidocking site located in the cytoplasm domain, resulting in theactivation of several key signalling pathways, including Ras-MAPK, PI3K,Src and Stat3 [Gao C F, Vande Woude G F, Cell Res. 2005, 15(1):49-51;Furge K A, Zhang Y W, Vande Woude G F, Oncogene. 2000, 19(49):5582-9].These pathways are essential for tumour cell proliferation, invasion andangiogenesis and for evading apoptosis [Furge K A, Zhang Y W, VandeWoude G F, Oncogene, 2000, 19(49):5582-9; Gu H, Neel B G, Trends CellBiol. 2003 March, 13(3):122-30; Fan S, Ma Y X, Wang J A, Yuan R Q, MengQ, Cao Y, Laterra J J, Goldberg I D, Rosen E M, Oncogene. 2000 April 27,19(18):2212-23]. In addition, a unique facet of the c-Met signallingrelative to other RTK is its reported interaction with focal adhesioncomplexes and non kinase binding partners such as α6β4 integrins[Trusolino L, Bertotti A, Comoglio P M, Cell. 2001, 107:643-54], CD44v6[Van der Voort R, Taher T E, Wielenga V J, Spaargaren M, Prevo R, SmitL, David G, Hartmann G, Gherardi E, Pals S T, J Biol. Chem. 1999,274(10):6499-506], Plexin B1 or semaphorins [Giordano S, Corso S,Conrotto P, Artigiani S, Gilestro G, Barberis D, Tamagnone L, Comoglio PM, Nat Cell Biol. 2002, 4(9):720-4; Conrotto P, Valdembri D, Corso S,Serini G, Tamagnone L, Comoglio P M, Bussolino F, Giordano S, Blood.2005, 105(11):4321-9; Conrotto P, Corso S, Gamberini S, Comoglio P M,Giordano S, Oncogene. 2004, 23:5131-7] which may further add to thecomplexity of regulation of cell function by this receptor. Finallyrecent data demonstrate that c-Met could be involved in tumor resistanceto gefitinib or erlotinib suggesting that combination of compoundtargeting both EGFR and c-Met might be of significant interest [EngelmanJ A at al., Science, 2007, 316:1039-43].

In the past few years, many different strategies have been developed toattenuate c-Met signalling in cancer cell lines. These strategiesinclude i) neutralizing antibodies against c-Met or HGF/SF [Cao B, Su Y,Oskarsson M, Zhao P, Kort E J, Fisher R J, Wang L M, Vande Woude G F,Proc Natl Acad Sci USA. 2001, 98(13):7443-8; Martens T, Schmidt N O,Eckerich C, Fillbrandt R, Merchant M, Schwall R, Westphal M, Lamszus K,Clin Cancer Res. 2006, 12(20):6144-52] or the use of HGF/SF antagonistNK4 to prevent ligand binding to c-Met [Kuba K, Matsumoto K, Date K,Shimura H, Tanaka M, Nakamura T, Cancer Res., 2000, 60:6737-43], ii)small ATP binding site inhibitors to c-Met that block kinase activity[Christensen J G, Schreck R, Burrows J, Kuruganti P, Chan E, Le P, ChenJ, Wang X, Ruslim L, Blake R, Lipson K E, Ramphal J, Do S, Cui J J,Chemington J M, Mendel D B, Cancer Res. 2003, 63:7345-55], iii)engineered SH2 domain polypeptide that interferes with access to themultidocking site and RNAi or ribozyme that reduce receptor or ligandexpression. Most of these approaches display a selective inhibition ofc-Met resulting in tumor inhibition and showing that c-Met could be ofinterest for therapeutic intervention in cancer.

Within the molecules generated for c-Met targeting, some are antibodies.

The most extensively described is the anti-c-Met 5D5 antibody generatedby Genentech [WO96/38557] which behaves as a potent agonist when addedalone in various models and as an antagonist when used as a Fabfragment. A monovalent engineered form of this antibody described as onearmed 5D5 (OA5D5) and produced as a recombinant protein in E. Coli isalso the subject of a patent application [WO2006/015371] by Genentech.However, this molecule that could not be considered as an antibodybecause of its particular scarfold, displays also mutations that couldbe immunogenic in humans. In terms of activity, this unglycosylatedmolecule is devoided of effector functions and finally, no clear datademonstrate that OA5D5 inhibits dimerization of c-Met. Moreover, whentested in the G55 in vivo model, a glioblastoma cell line that expressesc-Met but not HGF mRNA and protein and that grows independently of theligand, the one armed anti-c-Met had no significant effect on G55 tumorgrowth suggesting that OA5D5 acts primarily by blocking HGF binding andis not able to target tumors activated independently of HGF [Martens T.et al, Clin. Cancer Res., 2006, 12(20):6144-6152].

Another antibody targeting c-Met is described by Pfizer as an antibodyacting “predominantly as c-Met antagonist, and in some instance as ac-Met agonist” [WO 2005/016382]. No data showing any effect of Pfizerantibodies on c-Met dimerization is described in this application.

One of the innovant aspects of the present invention is to generatemouse monoclonal antibodies without intrinsic agonist activity andinhibiting c-Met dimerization. In addition of targeting ligand-dependenttumors, this approach will also impair ligand-independent activations ofc-Met due to its overexpression or mutations of the intra cellulardomains which remained dependent to oligomerization for signalling.Another aspect of the activity of such antibodies could be a sterichindrance for c-Met interaction with its partners that will result inimpairment of c-Met functions. These antibodies will be humanized andengineered preferentially, but not limited, as human IgG1 to geteffector functions such as ADCC and CDC in addition to functions linkedto the specific blockade of the c-Met receptor.

DISCLOSURE OF THE INVENTION

Surprisingly, for the first time, inventors have managed to generate anantibody capable of binding to c-Met but also capable of inhibiting thec-Met dimerization. If it is true that, in the prior art, it issometimes suggested that an antibody capable of inhibiting thedimerization of c-Met with its partners could be an interesting one, ithas never been disclosed, or clearly suggested, an antibody capable ofdoing so. Moreover, regarding antibody specificity, it was not evidentat all to succeed in the generation of such an active antibody.

In a first aspect, a subject of the present invention is a process forthe generation and the selection of antibodies according to theinvention.

More particularly, the invention concerns a process for the selection ofan anti c-Met antibody, or one of its functional fragments orderivatives, capable to inhibit both ligand-dependent andligand-independent activation of c-Met, said process comprising thefollowing steps:

i) screening the generated antibodies and selecting antibodies capableto bind specifically to c-Met;

ii) evaluating in vitro the selected antibodies of step i) and selectingantibodies capable to inhibit at least 50%, preferably at least 60%, 70%or 80% of tumoral cell proliferation for at least one tumor type; andthen

iii) testing the selected antibodies of step ii) and selectingantibodies capable to inhibit the c-Met dimerization.

As it was explained before, the inhibition of the c-Met dimerization isa capital aspect of the invention as such antibodies will present a realinterest for a larger population of patients. Not only ligand-dependentactivated c-Met cancer, as it was the case until the present invention,but also ligand-independent activated c-Met cancer could be treated withantibodies generated by the process of the present invention.

The generation of the antibody can be realized by any method known bythe man skilled in the art, such as for example, fusion of a myelomacell with spleen cells from immunized mice or other species compatiblewith the selected myeloma cells [Kohler & Milstein, 1975, Nature,256:495-497]. The immunized animals could include transgenic mice withhuman immunoglobulin loci which then directly produce human antibodies.Another possible embodiment could consist in using phage displaytechnologies to screen libraries.

The screening step i) can be realized by any method or process known bythe man skilled in the art. As non limitative examples, can be mentionedELISA, BIAcore, immunohistochemistry, FACS analysis and functionalscreens. A preferred process consists in a screen by ELISA on the c-Metrecombinant protein and then by FACS analysis on at least a tumoral cellline to be sure that the produced antibodies will be able to alsorecognize the native receptor on tumor cells. This process will bedescribed more precisely in the following examples.

In the same way, the step ii) can also be realized classically by knownmethod or process such as, for example, using 3H-thymidine or any otherDNA staining agent, MTT, ATP evaluation, etc. A preferred tumor cellmodel in the present invention can consist in the BxPC3 model.

By inhibiting c-Met dimerization, it must be understood preferably thec-Met homodimerization.

In a preferred embodiment of the step iii) of selection of the processof the invention, said step iii) consists in evaluating antibodies byBRET analysis on cells expressing both c-Met-RLuc/c-Met-YFP andselecting antibodies capable to inhibit at least 30%, preferably 35%,40%, 45%, 50%, 55% and most preferably 60% of the BRET signal.

The technology BRET is a technology known as being representative of theprotein dimerization [Angers et al., PNAS, 2000, 97:3684-89].

The technology BRET, used in the step iii) of the process, is well knownby the man skill in the art and will be detailed in the followingexamples. More particularly, BRET (Bioluminescence Resonance EnergyTransfer) is a non-radiative energy transfer occurring between abioluminescent donor (Renilla Luciferase (Rluc)) and a fluorescentacceptor, a mutant of GFP (Green Fluorescent Protein) or YFP (Yellowfluorescent protein). In the present case EYFP (Enhanced YellowFluorescent Protein) was used. The efficacy of transfer depends on theorientation and the distance between the donor and the acceptor. Then,the energy transfer can occur only if the two molecules are in closeproximity (1-10 nm). This property is used to generate protein-proteininteraction assays. Indeed, in order to study the interaction betweentwo partners, the first one is genetically fused to the RenillaLuciferase and the second one to the yellow mutant of the GFP. Fusionproteins are generally, but not obligatory, expressed in mammaliancells. In presence of its membrane permeable substrate (coelenterazine),Rluc emits blue light. If the GFP mutant is closer than 10 nm from theRluc, an energy transfer can occur and an additional yellow signal canbe detected. The BRET signal is measured as the ratio between the lightemitted by the acceptor and the light emitted by the donor. So the BRETsignal will increase as the two fusion proteins are brought intoproximity or if a conformational change bring Rluc and GFP mutantcloser.

If the BRET analysis consists in a preferred embodiment, any methodknown by the man skilled in the art can be used to measure c-Metdimerization. Without limitation, the following technologies can bementioned: FRET (Fluorescence Resonance Energy Transfer), HTRF(Homogenous Time resolved Fluorescence), FLIM (Fluorescence LifetimeImaging Microscopy) or SW-FCCS single wavelength fluorescencecross-correlation spectroscopy).

Other classical technologies could also be used, such asCo-immunoprecipitation, Alpha screen, Chemical cross-linking,Double-Hybrid, Affinity Chromatography, ELISA or Far western blot.

In a second aspect, a subject of the invention is an isolated antibody,or one of its functional fragments or derivatives, being obtained bysaid process. Said antibody or one of its said fragments or derivatives,is capable of binding specifically to the human c-Met and, if necessary,preferably moreover capable of inhibiting the natural attachment of itsligand HGF and/or capable of specifically inhibiting the tyrosine kinaseactivity of said c-Met, said antibody being also capable to inhib c-Metdimerization. More particularly, said antibodies will be capable ofinhibiting both ligand-dependent and ligand-independent activation ofc-Met.

The expressions “functional fragments and derivatives” will be definedin details later in the present specification.

It must be understood here that the invention does not relate to theantibodies in natural form, that is to say they are not in their naturalenvironment but that they have been able to be isolated or obtained bypurification from natural sources, or else obtained by geneticrecombination, or by chemical synthesis, and that they can then containunnatural amino acids as will be described further on.

More particularly, according to another aspect of the invention, it isclaimed an antibody, or one of its functional fragments or derivatives,said antibody being characterized in that it comprises at least onecomplementary determining region CDR chosen from CDRs comprising theamino acid sequence SEQ ID Nos. 1 to 17 and 56 to 61.

Any antibody, or fragments or derivatives, having at least one CDR whosesequence has at least 80% identity, preferably 85%, 90%, 95% and 98%identity, after optimum alignment with the sequences SEQ ID Nos. 1 to 17and 56 to 61 must be understood as a equivalent and, as a consequence,as being part of the invention.

By CDR regions or CDR(s), it is intended to indicate the hypervariableregions of the heavy and light chains of the immunoglobulins as definedby IMGT.

The IMGT unique numbering has been defined to compare the variabledomains whatever the antigen receptor, the chain type, or the species[Lefranc M.-P., Immunology Today 18, 509 (1997)/Lefranc M.-P., TheImmunologist, 7, 132-136 (1999)/Lefranc, M.-P., Pommié, C., Ruiz, M.,Giudicelli, V., Foulquier, E., Truong, L., Thouvenin-Contet, V. andLefranc, Dev. Comp. Immunol., 27, 55-77 (2003)]. In the IMGT uniquenumbering, the conserved amino acids always have the same position, forinstance cysteine 23 (1st-CYS), tryptophan 41 (CONSERVED-TRP),hydrophobic amino acid 89, cysteine 104 (2nd-CYS), phenylalanine ortryptophan 118 (J-PHE or J-TRP). The IMGT unique numbering provides astandardized delimitation of the framework regions (FR1-IMGT: positions1 to 26, FR2-IMGT: 39 to 55, FR3-IMGT: 66 to 104 and FR4-IMGT: 118 to128) and of the complementarity determining regions: CDR1-IMGT: 27 to38, CDR2-IMGT: 56 to 65 and CDR3-IMGT: 105 to 117. As gaps representunoccupied positions, the CDR-IMGT lengths (shown between brackets andseparated by dots, e.g. [8.8.13]) become crucial information. The IMGTunique numbering is used in 2D graphical representations, designated asIMGT Colliers de Perles [Ruiz, M. and Lefranc, M.-P., Immunogenetics,53, 857-883 (2002)/Kaas, Q. and Lefranc, M.-P., Current Bioinformatics,2, 21-30 (2007)], and in 3D structures in IMGT/3Dstructure-DB [Kaas, Q.,Ruiz, M. and Lefranc, M.-P., T cell receptor and MHC structural data.Nucl. Acids. Res., 32, D208-D210 (2004)].

Three heavy chain CDRs and 3 light chain CDRs exist. The term CDR orCDRs is used here in order to indicate, according to the case, one ofthese regions or several, or even the whole, of these regions whichcontain the majority of the amino acid residues responsible for thebinding by affinity of the antibody for the antigen or the epitope whichit recognizes.

By “percentage of identity” between two nucleic acid or amino acidsequences in the sense of the present invention, it is intended toindicate a percentage of nucleotides or of identical amino acid residuesbetween the two sequences to be compared, obtained after the bestalignment (optimum alignment), this percentage being purely statisticaland the differences between the two sequences being distributed randomlyand over their entire length. The comparisons of sequences between twonucleic acid or amino acid sequences are traditionally carried out bycomparing these sequences after having aligned them in an optimummanner, said comparison being able to be carried out by segment or by“comparison window”. The optimum alignment of the sequences for thecomparison can be carried out, in addition to manually, by means of thelocal homology algorithm of Smith and Waterman (1981) [Ad. App. Math.2:482], by means of the local homology algorithm of Neddleman and Wunsch(1970) [J. Mol. Biol. 48: 443], by means of the similarity search methodof Pearson and Lipman (1988) [Proc. Natl. Acad. Sci. USA 85:2444), bymeans of computer software using these algorithms (GAP, BESTFIT, FASTAand TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis., or else by BLAST N or BLAST Pcomparison software).

The percentage of identity between two nucleic acid or amino acidsequences is determined by comparing these two sequences aligned in anoptimum manner and in which the nucleic acid or amino acid sequence tobe compared can comprise additions or deletions with respect to thereference sequence for an optimum alignment between these two sequences.The percentage of identity is calculated by determining the number ofidentical positions for which the nucleotide or the amino acid residueis identical between the two sequences, by dividing this number ofidentical positions by the total number of positions in the comparisonwindow and by multiplying the result obtained by 100 in order to obtainthe percentage of identity between these two sequences.

For example, it is possible to use the BLAST program, “BLAST 2sequences” (Tatusova et al., “Blast 2 sequences—a new tool for comparingprotein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250)available on the site http://www.ncbi.nlm.nih.gov/gorf/b12.html, theparameters used being those given by default (in particular for theparameters “open gap penalty”: 5, and “extension gap penalty”: 2; thematrix chosen being, for example, the matrix “BLOSUM 62” proposed by theprogram), the percentage of identity between the two sequences to becompared being calculated directly by the program.

By amino acid sequence having at least 80%, preferably 85%, 90%, 95% and98% identity with a reference amino acid sequence, those having, withrespect to the reference sequence, certain modifications, in particulara deletion, addition or substitution of at least one amino acid, atruncation or an elongation are preferred. In the case of a substitutionof one or more consecutive or nonconsecutive amino acid(s), thesubstitutions are preferred in which the substituted amino acids arereplaced by “equivalent” amino acids. The expression “equivalent aminoacids” is aimed here at indicating any amino acid capable of beingsubstituted with one of the amino acids of the base structure without,however, essentially modifying the biological activities of thecorresponding antibodies and such as will be defined later, especiallyin the examples. These equivalent amino acids can be determined eitherby relying on their structural homology with the amino acids which theyreplace, or on results of comparative trials of biological activitybetween the different antibodies capable of being carried out.

By way of example, mention is made of the possibilities of substitutioncapable of being carried out without resulting in a profoundmodification of the biological activity of the corresponding modifiedantibody.

As non limitative example, the following table 1 is giving substitutionpossibilities conceivable with a conservation of the biological activityof the modified antibody. The reverse substitutions are also, of course,possible in the same conditions.

TABLE 1 Original residu Substitution(s) Ala (A) Val, Gly, Pro Arg (R)Lys, His Asn (N) Gln Asp (D) Glu Cys (C) Ser Gln (Q) Asn Glu (G) Asp Gly(G) Ala His (H) Arg Ile (I) Leu Leu (L) Ile, Val, Met Lys (K) Arg Met(M) Leu Phe (F) Tyr Pro (P) Ala Ser (S) Thr, Cys Thr (T) Ser Trp (W) TyrTyr (Y) Phe, Trp Val (V) Leu, Ala

It must be understood here that the invention does not relate to theantibodies in natural form, that is to say they are not in their naturalenvironment but that they have been able to be isolated or obtained bypurification from natural sources, or else obtained by geneticrecombination, or by chemical synthesis, and that they can then containunnatural amino acids as will be described further on.

According a first approach, the antibody will be defined by its heavychain sequence. More particularly, the antibody of the invention, or oneof its functional fragments or derivatives, is characterized in that itcomprises a heavy chain comprising at least one CDR chosen from CDRscomprising the amino acid sequences SEQ ID Nos. 1 to 9 and 56 to 58.

The mentioned sequences are the following ones:

SEQ ID No. 1: GYIFTAYT SEQ ID No. 2: IKPNNGLA SEQ ID No. 3: ARSEITTEFDYSEQ ID No. 4: GYSFTDYT SEQ ID No. 5: INPYNGGT SEQ ID No. 6: AREEITKDFDFSEQ ID No. 7: GYTFTDYN SEQ ID No. 8: INPNNGGT SEQ ID No. 9:ARGRYVGYYYAMDY SEQ ID No. 56: GYTFTSYW SEQ ID No. 57: INPTTGSTSEQ ID No. 58: AIGGYGSWFAY

The CDRs of the heavy chain could be chosen randomly in the previoussequences, i.e. SEQ ID Nos. 1 to 9 and 56 to 58.

According to a preferred aspect, the antibody of the invention, or oneof its functional fragments or derivatives, comprises a heavy chaincomprising at least one CDR chosen from CDR-H1, CDR-H2 and CDR-H3,wherein:

-   -   CDR-H1 comprises the amino acid sequence SEQ ID No. 1, 4, 7 or        56,    -   CDR-H2 comprises the amino acid sequence SEQ ID No. 2, 5, 8 or        57, and    -   CDR-H3 comprises the amino acid sequence SEQ ID No. 3, 6, 9 or        58.

According to a first embodiment of said aspect, the antibody of theinvention, or one of its functional fragments or derivatives, comprisesa heavy chain comprising CDR-H1, CDR-H2 and CDR-H3, wherein CDR-H1comprises the amino acid sequence SEQ ID No. 1, CDR-H2 comprises theamino acid sequence SEQ ID No. 2 and CDR-H3 comprises the amino acidsequence SEQ ID No. 3.

More particularly, said antibody, or one of its functional fragments orderivatives, according to this first embodiment comprises a heavy chainof sequence comprising the amino acid sequence SEQ ID No. 18.

SEQ ID No. 18: EVQLQQSGPELVKPGASVKISCKTSGYIFTAYTMHWVRQSLGESLDWIGGIKPNNGLANYNQKFKGKATLTVDKSSSTAYMDLRSLTSEDSAVYYCARSEITTEFDYWGQGTALTVSS

According to a second embodiment of said aspect, the antibody of theinvention, or one of its functional fragments or derivatives, comprisesa heavy chain comprising CDR-H1, CDR-H2 and CDR-H3, wherein CDR-H1comprises the amino acid sequence SEQ ID No. 4, CDR-H2 comprises theamino acid sequence SEQ ID No. 5 and CDR-H3 comprises the amino acidsequence SEQ ID No. 6.

The antibody, or one of its functional fragments or derivatives,according to said second embodiment will preferably comprise a heavychain of sequence comprising the amino acid sequence SEQ ID No. 19.

SEQ ID No. 19: EVQLQQSGPELVKPGASMKISCKASGYSFTDYTLNWVKQSHGKTLEWIGLINPYNGGTTYNQKFKGKATLTVDKSSSTAYMELLSLTSEDSAVYYCAREEITKDFDFWGQGTTLTVSS

According to a third embodiment of said aspect, the antibody of theinvention, or one of its functional fragments or derivatives, comprisesa heavy chain comprising CDR-H1, CDR-H2 and CDR-H3, wherein CDR-H1comprises the amino acid sequence SEQ ID No. 7, CDR-H2 comprises theamino acid sequence SEQ ID No. 8 and CDR-H3 comprises the amino acidsequence SEQ ID No. 9.

The antibody, or one of its functional fragments or derivatives,according to said third embodiment will preferably comprise a heavychain of sequence comprising the amino acid sequence SEQ ID No. 20.

SEQ ID No. 20: EVLLQQSGPELVKPGASVKIPCKASGYTFTDYNMDWVKQSHGMSLEWIGDINPNNGGTIFNQKFKGKATLTVDKSSSTAYMELRSLTSEDTAVYYCARGRYVGYYYAMDYWGQGTSVTVSS

According to a fourth embodiment of said aspect, the antibody of theinvention, or one of its functional fragments or derivatives, comprisesa heavy chain comprising CDR-H1, CDR-H2 and CDR-H3, wherein CDR-H1comprises the amino acid sequence SEQ ID No. 56, CDR-H2 comprises theamino acid sequence SEQ ID No. 57 and CDR-H3 comprises the amino acidsequence SEQ ID No. 58.

The antibody, or one of its functional fragments or derivatives,according to said fourth embodiment will preferably comprise a heavychain of sequence comprising the amino acid sequence SEQ ID No. 62.

SEQ ID No. 62: QVQLQQSGAELAKPGASVKMSCKASGYTFTSYWMNWVKQRPGQGLEWIGYINPTTGSTDYNQKLKDKATLTADKSSNTAYMQLSSLTSEDSAVYYCAI GGYGSWFAYWGQGTLVTVSA

In a second approach, the antibody will be now define by its light chainsequence. More particularly, according to a second particular aspect ofthe invention, the antibody, or one of its functional fragments orderivatives, is characterized in that it comprises a light chaincomprising at least one CDR chosen from CDRs comprising the amino acidsequence SEQ ID Nos. 10 to 17 and 59 to 61.

The mentioned sequences are the following ones:

SEQ ID No. 10: ESVDSYANSF SEQ ID No. 11: RAS SEQ ID No. 12: QQSKEDPLTSEQ ID No. 13: ESIDTYGNSF SEQ ID No. 14: QQSNEDPFT SEQ ID No. 15: ENIYSNSEQ ID No. 16: AAT SEQ ID No. 17: QHFWGPPYT SEQ ID No. 59: SSVSSTYSEQ ID No. 60: TTS SEQ ID No. 61: HQWSSYPFT

The CDRs of the light chain could be chosen randomly in the previoussequences, i.e. SEQ ID Nos. 10 to 17 and 59 to 61.

According to another preferred aspect, the antibody of the invention, orone of its functional fragments or derivatives, comprises a light chaincomprising at least one CDR chosen from CDR-L1, CDR-L2 and CDR-L3,wherein:

-   -   CDR-L1 comprises the amino acid sequence SEQ ID No. 10, 13, 15        or 59,    -   CDR-L2 comprises the amino acid sequence SEQ ID No. 11, 16 or        60, and    -   CDR-L3 comprises the amino acid sequence SEQ ID No. 12, 14, 17        or 61.

According to a first embodiment of said another aspect, the antibody ofthe invention, or one of its functional fragments or derivatives,comprises a light chain comprising CDR-L1, CDR-L2 and CDR-L3, whereinCDR-L1 comprises the amino acid sequence SEQ ID No. 10, CDR-L2 comprisesthe amino acid sequence SEQ ID No. 11 and CDR-L3 comprises the aminoacid sequence SEQ ID No. 12.

More particularly, said antibody, or one of its functional fragments orderivatives, according to this first embodiment comprises a light chainof sequence comprising the amino acid sequence SEQ ID No. 21.

SEQ ID No. 21: DIVLTQSPASLAVSLGQRATISCRASESVDSYANSFMHWYQQKPGQPPKLLIYRASNLESGIPARFSGSGSRTDFTLTINPVEADDVATYY CQQSKEDPLTFGSGTKLEMK

According to a second embodiment of said another aspect, the antibody ofthe invention, or one of its functional fragments or derivatives,comprises a light chain comprising CDR-L1, CDR-L2 and CDR-L3, whereinCDR-L1 comprises the amino acid sequence SEQ ID No. 13, CDR-L2 comprisesthe amino acid sequence SEQ ID No. 11 and CDR-L3 comprises the aminoacid sequence SEQ ID No. 14.

The antibody, or one of its functional fragments or derivatives,according to said second embodiment will preferably comprise a lightchain of sequence comprising the amino acid sequence SEQ ID No. 22.

SEQ ID No. 22: GIVLTQSPASLAVSLGQRATISCRVSESIDTYGNSFIHWYQQKPGQPPKLLIYRASNLESGIPARFSGSGSRTDFTLTINPVEADDSATYYCQ QSNEDPFTFGSGTKLEMK

According to a third embodiment of said another aspect, the antibody ofthe invention, or one of its functional fragments or derivatives,comprises a light chain comprising CDR-L1, CDR-L2 and CDR-L3, whereinCDR-L1 comprises the amino acid sequence SEQ ID No. 15, CDR-L2 comprisesthe amino acid sequence SEQ ID No. 16 and CDR-L3 comprises the aminoacid sequence SEQ ID No. 17.

The antibody, or one of its functional fragments or derivatives,according to said third embodiment will preferably comprise a lightchain of sequence comprising the amino acid sequence SEQ ID No. 23.

SEQ ID No. 23: DIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGKSPQLLVYAATNLVDGVPSRFSGSGSGTQYSLKINSLQSEDFGSYYCQHFWG PPYTFGGGTKLEIK

According to a fourth embodiment of said another aspect, the antibody ofthe invention, or one of its functional fragments or derivatives,comprises a light chain comprising CDR-L1, CDR-L2 and CDR-L3, whereinCDR-L1 comprises the amino acid sequence SEQ ID No. 59, CDR-L2 comprisesthe amino acid sequence SEQ ID No. 60 and CDR-L3 comprises the aminoacid sequence SEQ ID No. 61.

The antibody, or one of its functional fragments or derivatives,according to said third embodiment will preferably comprise a lightchain of sequence comprising the amino acid sequence SEQ ID No. 63.

SEQ ID No. 63: QIVLTQSPAIMSASPGEKVTLTCSASSSVSSTYLYWYQQKPGSSPKLWIYTTSILASGVPARFSGSGSGTSYSLTISSMETEDAASYFCHQWSSYPFT FGSGTKLDIK

According a third approach, the antibody will be now defined both by itslight chain sequence and its heavy chain sequence. The antibody of theinvention, or one of its functional fragments or derivatives, ischaracterized in that it comprises a heavy chain comprising the aminoacid sequence SEQ ID No. 18, 19, 20 or 62 and a light chain comprisingthe amino acid sequence SEQ ID No. 21, 22, 23 or 63.

More particularly, a preferred antibody, or one of its functionalfragments or derivatives, according to the invention, named 224G11,comprises a heavy chain comprising CDR-H1, CDR-H2 and CDR-H3 comprisingrespectively the amino acid sequence SEQ ID Nos. 1, 2 and 3; and a lightchain comprising CDR-L1, CDR-L2 and CDR-L3 comprising respectively theamino acid sequence SEQ ID Nos. 10, 11 and 12.

In another aspect, the antibody 224G11 comprises a heavy chaincomprising the amino acid sequence SEQ ID No. 18 and a light chaincomprising the amino acid sequence SEQ ID No. 21.

Another preferred antibody, or one of its functional fragments orderivatives, according to the invention, named 227H1, comprises a heavychain comprising CDR-H1, CDR-H2 and CDR-H3 comprising respectively theamino acid sequence SEQ ID Nos. 4, 5 and 6; and a light chain comprisingCDR-L1, CDR-L2 and CDR-L3 comprising respectively the amino acidsequence SEQ ID Nos. 13, 11 and 14.

In another aspect, the antibody 227H1 comprises a heavy chain comprisingthe amino acid sequence SEQ ID No. 19 and a light chain comprising theamino acid sequence SEQ ID No. 22.

Still another preferred antibody, or one of its functional fragments orderivatives, named 223C4, comprises a heavy chain comprising CDR-H1,CDR-H2 and CDR-H3 comprising respectively the amino acid sequence SEQ IDNos. 7, 8 and 9; and a light chain comprising CDR-L1, CDR-L2 and CDR-L3comprising respectively the amino acid sequence SEQ ID Nos. 15, 16 and17.

In another aspect, the antibody 223C4 comprises a heavy chain comprisingthe amino acid sequence SEQ ID No. 20 and a light chain comprising theamino acid sequence SEQ ID No. 23.

Still another preferred antibody, or one of its functional fragments orderivatives, named 11E1, comprises a heavy chain comprising CDR-H1,CDR-H2 and CDR-H3 comprising respectively the amino acid sequence SEQ IDNos. 56, 57 and 58; and a light chain comprising CDR-L1, CDR-L2 andCDR-L3 comprising respectively the amino acid sequence SEQ ID Nos. 59,60 and 61.

In another aspect, the antibody 11E1 comprises a heavy chain comprisingthe amino acid sequence SEQ ID No. 62 and a light chain comprising theamino acid sequence SEQ ID No. 63.

According to another aspect, the invention relates to murine hybridomacapable of secreting monoclonal antibodies according to the presentinvention, especially hybridoma of murine origin such as deposited atthe Collection Nationale de Cultures de Microorganismes (CNCM, NationalCollection of Microorganism Cultures) (Institut Pasteur, Paris, France).

The monoclonal antibodies according to the invention, or one of theirfunctional fragments or derivatives, are characterized in that saidantibodies are secreted by the hybridoma deposited at the CNCM on Mar.14, 2007 under the numbers CNCM 1-3724 (corresponding to 11E1), 1-3731(corresponding to 224G11), 1-3732 (corresponding to 227H1) and on Jul.6, 2007 under the number 1-3786 (corresponding to 223C4). Thesehybridoma consist in murine hybridoma resulting in the cellular fusionof immunized mouse splenocytes with a myeloma cell line (Sp20 Ag14).

The following table 2 regroups elements concerning the preferredantibodies.

TABLE 2 224G11 227H1 223C4 11E1 I-3731 I-3732 I-3786 I-3724 Prot. Nucl.Prot; Nucl. Prot. Nucl. Prot. Nucl. SEQ ID SEQ ID SEQ ID SEQ ID SEQ IDSEQ ID SEQ ID SEQ ID CDR-H1 1 24 4 27 7 30 56 64 CDR-H2 2 25 5 28 8 3157 65 CDR-H3 3 26 6 29 9 32 58 66 H. chain 18 41 19 42 20 43 62 70CDR-L1 10 33 13 36 15 38 59 67 CDR-L2 11 34 11 34 16 39 60 68 CDR-L3 1235 14 37 17 40 61 69 L. chain 21 44 22 45 23 46 63 71

From table 2, it clearly appears that CDR-L2 of the antibodies 227H1 and224G11 is similar. This example clearly supports the claims of thepresent application covering antibodies comprising at least one CDRrandomly chosen through described CDR sequences.

According to a preferred embodiment, the invention relates to monoclonalantibodies.

The term <<Monoclonal Antibody>> or is used in accordance with itsordinary meaning to denote an antibody obtained from a population ofsubstantially homogeneous antibodies, i.e. the individual antibodiescomprising the population are identical except for possible naturallyoccurring mutations that may be present in minor amounts. In otherwords, a monoclonal antibody consists in a homogenous antibody resultingfrom the proliferation of a single clone of cells (e.g., hybridomacells, eukaryotic host cells transfected with DNA encoding thehomogenous antibody, prokaryotic host cells transformed with DNAencoding the homogenous antibody, etc.), and which is generallycharacterized by heavy chains of a single class and subclass, and lightchains of a single type. Monoclonal antibodies are highly specific,being directed against a single antigen. Furthermore, in contrast topolyclonal antibodies preparations that typically include differentantibodies directed against different determinants, or epitope, eachmonoclonal antibody is directed against a single determinant on theantigen.

In the present description, the terms polypeptides, polypeptidesequences, amino acid sequences, peptides and proteins attached toantibody compounds or to their sequence are interchangeable.

According to a likewise particular aspect, the present invention relatesto a chimeric antibody, or one of its functional fragments, according tothe invention, characterized in that said antibody moreover comprisesthe light chain and heavy chain constant regions derived from anantibody of a species heterologous to the mouse, especially man, and ina preferred manner in that the light chain and heavy chain constantregions derived from a human antibody are respectively the kappa andgamma-1, gamma-2 or gamma-4 region.

In the present application, IgG1 are preferred to get effectorfunctions, and most preferably ADCC and CDC.

The skilled artisan will recognize that effector functions include, forexample, Clq binding; complement dependent cytotoxicity (CDC); Fcreceptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC);phagocytosis; and down regulation of cell surface receptors (e.g. B cellreceptor; BCR).

The antibodies according to the present invention, are preferablyspecific monoclonal antibodies, especially of murine, chimeric orhumanized origin, which can be obtained according to the standardmethods well known to the person skilled in the art.

In general, for the preparation of monoclonal antibodies or theirfunctional fragments or derivatives, especially of murine origin, it ispossible to refer to techniques which are described in particular in themanual “Antibodies” (Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory, Cold Spring Harbor N.Y., pp. 726, 1988)or to the technique of preparation from hybridomas described by Kohlerand Milstein (Nature, 256:495-497, 1975).

The monoclonal antibodies according to the invention can be obtained,for example, from an animal cell immunized against the c-Met, or one ofits fragments containing the epitope specifically recognized by saidmonoclonal antibodies according to the invention. Said c-Met, or one ofits said fragments, can especially be produced according to the usualworking methods, by genetic recombination starting with a nucleic acidsequence contained in the cDNA sequence coding for the c-Met or bypeptide synthesis starting from a sequence of amino acids comprised inthe peptide sequence of the c-Met.

The monoclonal antibodies according to the invention can, for example,be purified on an affinity column on which the c-Met or one of itsfragments containing the epitope specifically recognized by saidmonoclonal antibodies according to the invention has previously beenimmobilized. More particularly, said monoclonal antibodies can bepurified by chromatography on protein A and/or G, followed or notfollowed by ion-exchange chromatography aimed at eliminating theresidual protein contaminants as well as the DNA and the LPS, in itselffollowed or not followed by exclusion chromatography on Sepharose™ gelin order to eliminate the potential aggregates due to the presence ofdimers or of other multimers. In an even more preferred manner, thewhole of these techniques can be used simultaneously or successively.

Chimeric or humanized antibodies are likewise included in antibodiesaccording to the present invention.

By chimeric antibody, it is intended to indicate an antibody whichcontains a natural variable (light chain and heavy chain) region derivedfrom an antibody of a given species in combination with the light chainand heavy chain constant regions of an antibody of a speciesheterologous to said given species (e.g. mouse, horse, rabbit, dog, cow,chicken, etc.).

The antibodies or their fragments of chimeric type according to theinvention can be prepared by using the techniques of geneticrecombination. For example, the chimeric antibody can be produced bycloning a recombinant DNA containing a promoter and a sequence codingfor the variable region of a non-human, especially murine, monoclonalantibody according to the invention and a sequence coding for theconstant region of human antibody. A chimeric antibody of the inventionencoded by such a recombinant gene will be, for example, a mouse-manchimera, the specificity of this antibody being determined by thevariable region derived from the murine DNA and its isotype determinedby the constant region derived from the human DNA. For the methods ofpreparation of chimeric antibodies, it is possible, for example, torefer to the documents Verhoeyn et al. (BioEssays, 8:74, 1988), Morrisonet al. (Proc. Natl. Acad. Sci. USA 82:6851-6855, 1984) ou le brevet U.S.Pat. No. 4,816,567.

By humanized antibody, it is intended to indicate an antibody whichcontains CDR regions derived from an antibody of nonhuman origin, theother parts of the antibody molecule being derived from one (or fromseveral) human antibodies. Moreover, some of the residues of thesegments of the skeleton (called FR) can be modified in order toconserve the affinity of the binding (Jones et al., Nature, 321:522-525,1986; Verhoeyen et al., Science, 239:1534-1536, 1988; Riechmann et al.,Nature, 332:323-327, 1988).

The humanized antibodies according to the invention or their fragmentscan be prepared by techniques known to the person skilled in the art(such as, for example, those described in the documents Singer et al.,J. Immun. 150:2844-2857, 1992; Mountain et al., Biotechnol. Genet. Eng.Rev., 10:1-142, 1992; or Bebbington et al., Bio/Technology, 10:169-175,1992).

Other humanization method are known by the man skill in the art as, forexample, the “CDR Grafting” method described by Protein Design Lab (PDL)in the patent applications EP 0 451216, EP 0 682 040, EP 0 939127, EP 0566 647 or U.S. Pat. No. 5,530,101, U.S. Pat. No. 6,180,370, U.S. Pat.No. 5,585,089 and U.S. Pat. No. 5,693,761. The following patentapplications can also be mentioned: U.S. Pat. No. 5,639,641; U.S. Pat.No. 6,054,297; U.S. Pat. No. 5,886,152 and U.S. Pat. No. 5,877,293.

By “functional fragment” of an antibody according to the invention, itis intended to indicate in particular an antibody fragment, such as Fv,scFv (sc for single chain), Fab, F(ab′)₂, Fab′, scFv-Fc fragments ordiabodies, or any fragment of which the half-life time would have beenincreased by chemical modification, such as the addition ofpoly(alkylene)glycol such as poly(ethylene)glycol (“PEGylation”)(pegylated fragments called Fv-PEG, scFv-PEG, Fab-PEG, F(ab′)₂—PEG orFab′-PEG) (“PEG” for Poly(Ethylene)Glycol), or by incorporation in aliposome, said fragments having at least one of the characteristic CDRsof sequence SEQ ID Nos. 1 to 17 and 56 to 61 according to the invention,and, especially, in that it is capable of exerting in a general manneran even partial activity of the antibody from which it is descended,such as in particular the capacity to recognize and to bind to thec-Met, and, if necessary, to inhibit the activity of the c-Met.

Preferably, said functional fragments will be constituted or willcomprise a partial sequence of the heavy or light variable chain of theantibody from which they are derived, said partial sequence beingsufficient to retain the same specificity of binding as the antibodyfrom which it is descended and a sufficient affinity, preferably atleast equal to 1/100, in a more preferred manner to at least 1/10, ofthat of the antibody from which it is descended, with respect to thec-Met. Such a functional fragment will contain at the minimum 5 aminoacids, preferably 6, 7, 8, 9, 10, 12, 15, 25, 50 and 100 consecutiveamino acids of the sequence of the antibody from which it is descended.

Preferably, these functional fragments will be fragments of Fv, scFv,Fab, F(ab′)₂, F(ab′), scFv-Fc type or diabodies, which generally havethe same specificity of binding as the antibody from which they aredescended. In a more preferred embodiment of the invention, thesefragments are selected among divalent fragments such as F(ab′)₂fragments. According to the present invention, antibody fragments of theinvention can be obtained starting from antibodies such as describedabove by methods such as digestion by enzymes, such as pepsin or papainand/or by cleavage of the disulfide bridges by chemical reduction. Inanother manner, the antibody fragments comprised in the presentinvention can be obtained by techniques of genetic recombinationlikewise well known to the person skilled in the art or else by peptidesynthesis by means of, for example, automatic peptide synthesizers suchas those supplied by the company Applied Biosystems, etc.

By “divalent fragment”, it must be understood any antibody fragmentscomprising two arms and, more particularly, F(ab′)₂ fragments.

More particularly, the invention comprises the antibodies, or theirfunctional fragments, according to the present invention, especiallychimeric or humanized antibodies, obtained by genetic recombination orby chemical synthesis.

By <<derivatives>> of an antibody according to the invention, it ismeant a binding protein comprising a protein scaffold and at least on ofthe CDRs selected from the original antibody in order to maintain thebinding capacity. Such compounds are well known by the man skilled inthe art and will be described in more details in the followingspecification.

More particularly, the antibody, or one of its functional fragments orderivatives, according to the invention is characterized in that sandderivative consists in a binding protein comprising a scaffold on whichat least one CDR has been grafted for the conservation of the originalantibody paratopic recognizing properties.

One or several sequences through the 6 CDR sequences described in theinvention can be presented on a protein scaffold. In this case, theprotein scaffold reproduces the protein backbone with appropriatefolding of the grafted CDR(s), thus allowing it (or them) to maintaintheir antigen paratopic recognizing properties. The man skilled in theart knows how to select the protein scaffold on which at least one CDRselected from the original antibody could be grafted. More particularly,it is known that, to be selected, such scaffold should display severalfeatures as follow (Skerra A., J. Mol. Recogn., 13, 2000, 167-187):

-   -   phylogenetically good conservation,    -   robust architecture with a well known three-dimensional        molecular organization (such as, for example, crystallography or        NMR),    -   small size,    -   no or only low degree of post-translational modifications,    -   easy to produce, express and purify.

Such protein scaffold can be, but without limitation, structure selectedfrom the group consisting in fibronectin and preferentially the tenthfibronectin type III domain (FNfn10), lipocalin, anticalin (Skerra A.,J. Biotechnol., 2001, 74(4):257-75), the protein Z derivative from thedomain B of staphylococcal protein A, thioredoxin A or any protein withrepeated domain such as “ankyrin repeat” (Kohl et al., PNAS, 2003, vol.100, No. 4, 1700-1705), “armadillo repeat”, “leucin-rich repeat” or“tetratricopeptide repeat”.

It could also be mentioned scaffold derivative from toxins (such as, forexample, scorpion, insect, plant or mollusc toxins) or proteininhibitors of neuronal nitric oxyde synthase (PIN).

As non limitative example of such hybrid constructions, it can bementioned the insertion of the CDR-H1 (heavy chain) of an anti-CD4antibody, i.e. the 13B8.2 antibody, into one of the exposed loop of thePIN. The binding properties of the obtained binding protein remainsimilar to the original antibody (Bes et al., BBRC 343, 2006, 334-344).It can also be mentioned the grafting of the CDR-H3 (heavy chain) of ananti-lyzozyme VHH antibody on a loop of neocarzinostatine (Nicaise etal., 2004).

In the case of the present invention, an interesting CDR to conservecould be, without limitation, the CDR-L2 as it is conserved in twoidentified antibodies of the invention, i.e. 227H1 and 224G11.

As above mentioned, such protein scaffold can comprise from 1 to 6CDR(s) from the original antibody. In a preferred embodiment, butwithout any limitation, the man skilled in the art would select at leasta CDR from the heavy chain, said heavy chain being known to beparticularly implicated in the antibody specificity. The selection ofthe CDR(s) of interest will be evident for the man of the art with knownmethod (BES et al., FEBS letters 508, 2001, 67-74).

As an evidence, these examples are not limitative and any other scaffoldknown or described must be included in the present specification.

According to a novel aspect, the present invention relates to anisolated nucleic acid, characterized in that it is chosen from thefollowing nucleic acids:

a) a nucleic acid, DNA or RNA, coding for an antibody, or one of itsfunctional fragments or derivatives, according to the invention;

b) a nucleic acid comprising a DNA sequence selecting from the group ofsequences consisting of:

-   -   a nucleic sequence comprising the sequences SEQ ID No. 24, SEQ        ID No. 25, SEQ ID No. 26 and the sequences SEQ ID No. 33, SEQ ID        No. 34 and SEQ ID No. 35;    -   a nucleic sequence comprising the sequences SEQ ID No. 27, SEQ        ID No. 28, SEQ ID No. 29 and the sequences SEQ ID No. 36, SEQ ID        No. 34 and SEQ ID No. 37;    -   a nucleic sequence comprising the sequences SEQ ID No. 30, SEQ        ID No. 31, SEQ ID No. 32 and the sequences SEQ ID No. 38, SEQ ID        No. 39 and SEQ ID No. 40; and    -   a nucleic sequence comprising the sequences SEQ ID No. 64, SEQ        ID No. 65, SEQ ID No. 66 and the sequences SEQ ID No. 67, SEQ ID        No. 68 and SEQ ID No. 69;

c) a nucleic acid comprising a DNA sequence selecting from the group ofsequences consisting of:

-   -   a nucleic sequence comprising the sequences SEQ ID No. 41 and        SEQ ID No. 44;    -   a nucleic sequence comprising the sequences SEQ ID No. 42 and        SEQ ID No. 45;    -   a nucleic sequence comprising the sequences SEQ ID No. 43 and        SEQ ID No. 46, and    -   a nucleic sequence comprising the sequences SEQ ID No. 70 and        SEQ ID No. 71;

d) the corresponding RNA nucleic acids of the nucleic acids as definedin b) or c);

e) the complementary nucleic acids of the nucleic acids as defined ina), b) and c); and

f) a nucleic acid of at least 18 nucleotides capable of hybridizingunder conditions of height stringency with at least one of the CDRs ofsequence SEQ ID Nos. 24 to 40 and 64 to 69.

By nucleic acid, nucleic or nucleic acid sequence, polynucleotide,oligonucleotide, polynucleotide sequence, nucleotide sequence, termswhich will be employed indifferently in the present invention, it isintended to indicate a precise linkage of nucleotides, which aremodified or unmodified, allowing a fragment or a region of a nucleicacid to be defined, containing or not containing unnatural nucleotides,and being able to correspond just as well to a double-stranded DNA, asingle-stranded DNA as to the transcription products of said DNAs.

It must also be understood here that the present invention does notconcern the nucleotide sequences in their natural chromosomalenvironment, that is to say in the natural state. It concerns sequenceswhich have been isolated and/or purified, that is to say that they havebeen selected directly or indirectly, for example by copy, theirenvironment having been at least partially modified. It is thus likewiseintended to indicate here the isolated nucleic acids obtained by geneticrecombination by means, for example, of host cells or obtained bychemical synthesis.

An hybridization under conditions of high stringency signifies that thetemperature conditions and ionic strength conditions are chosen in sucha way that they allow the maintenance of the hybridization between twofragments of complementary DNA. By way of illustration, conditions ofhigh stringency of the hybridization step for the purposes of definingthe polynucleotide fragments described above are advantageously thefollowing.

The DNA-DNA or DNA-RNA hybridization is carried out in two steps: (1)prehybridization at 42° C. for 3 hours in phosphate buffer (20 mM, pH7.5) containing 5×SSC (1×SSC corresponds to a 0.15 M NaCl+0.015 M sodiumcitrate solution), 50% of formamide, 7% of sodium dodecyl sulfate (SDS),10×Denhardt's, 5% of dextran sulfate and 1% of salmon sperm DNA; (2)actual hybridization for 20 hours at a temperature dependent on the sizeof the probe (i.e.: 42° C., for a probe size >100 nucleotides) followedby 2 washes of 20 minutes at 20° C. in 2×SSC+2% of SDS, 1 wash of 20minutes at 20° C. in 0.1×SSC+0.1% of SDS. The last wash is carried outin 0.1×SSC+0.1% of SDS for 30 minutes at 60° C. for a probe size >100nucleotides. The hybridization conditions of high stringency describedabove for a polynucleotide of defined size can be adapted by the personskilled in the art for oligonucleotides of greater or smaller size,according to the teaching of Sambrook et al. (1989, Molecular cloning: alaboratory manual. 2nd Ed. Cold Spring Harbor).

The invention likewise relates to a vector comprising a nucleic acidaccording to the present invention.

The invention aims especially at cloning and/or expression vectors whichcontain a nucleotide sequence according to the invention.

The vectors according to the invention preferably contain elements whichallow the expression and/or the secretion of the nucleotide sequences ina determined host cell. The vector must therefore contain a promoter,signals of initiation and termination of translation, as well asappropriate regions of regulation of transcription. It must be able tobe maintained in a stable manner in the host cell and can optionallyhave particular signals which specify the secretion of the translatedprotein. These different elements are chosen and optimized by the personskilled in the art as a function of the host cell used. To this effect,the nucleotide sequences according to the invention can be inserted intoautonomous replication vectors in the chosen host, or be integrativevectors of the chosen host.

Such vectors are prepared by methods currently used by the personskilled in the art, and the resulting clones can be introduced into anappropriate host by standard methods, such as lipofection,electroporation, thermal shock, or chemical methods.

The vectors according to the invention are, for example, vectors ofplasmidic or viral origin. They are useful for transforming host cellsin order to clone or to express the nucleotide sequences according tothe invention.

The invention likewise comprises the host cells transformed by orcomprising a vector according to the invention.

The host cell can be chosen from prokaryotic or eukaryotic systems, forexample bacterial cells but likewise yeast cells or animal cells, inparticular mammalian cells. It is likewise possible to use insect cellsor plant cells.

The invention likewise concerns animals, except man, which comprise atleast one cell transformed according to the invention.

According to another aspect, a subject of the invention is a process forproduction of an antibody, or one of its functional fragments accordingto the invention, characterized in that it comprises the followingstages:

a) culture in a medium and appropriate culture conditions of a host cellaccording to the invention; and

b) the recovery of said antibodies, or one of their functionalfragments, thus produced starting from the culture medium or saidcultured cells.

The cells transformed according to the invention can be used inprocesses for preparation of recombinant polypeptides according to theinvention. The processes for preparation of a polypeptide according tothe invention in recombinant form, characterized in that they employ avector and/or a cell transformed by a vector according to the invention,are themselves comprised in the present invention. Preferably, a celltransformed by a vector according to the invention is cultured underconditions which allow the expression of said polypeptide and saidrecombinant peptide is recovered.

As has been said, the host cell can be chosen from prokaryotic oreukaryotic systems. In particular, it is possible to identify nucleotidesequences according to the invention, facilitating secretion in such aprokaryotic or eukaryotic system. A vector according to the inventioncarrying such a sequence can therefore advantageously be used for theproduction of recombinant proteins, intended to be secreted. In effect,the purification of these recombinant proteins of interest will befacilitated by the fact that they are present in the supernatant of thecell culture rather than in the interior of the host cells.

It is likewise possible to prepare the polypeptides according to theinvention by chemical synthesis. Such a preparation process is likewisea subject of the invention. The person skilled in the art knows theprocesses of chemical synthesis, for example the techniques employingsolid phases [Steward et al., 1984, Solid phase peptide synthesis,Pierce Chem. Company, Rockford, 111, 2nd ed., (1984)] or techniquesusing partial solid phases, by condensation of fragments or by aclassical synthesis in solution. The polypeptides obtained by chemicalsynthesis and being able to contain corresponding unnatural amino acidsare likewise comprised in the invention.

The antibodies, or one of their functional fragments or derivatives,capable of being obtained by a process according to the invention arelikewise comprised in the present invention.

The invention also concerns the antibody of the invention as amedicament.

The invention likewise concerns a pharmaceutical composition comprisingby way of active principle a compound consisting of an antibody, or oneof its functional fragments according to the invention, preferably mixedwith an excipient and/or a pharmaceutically acceptable vehicle.

Another complementary embodiment of the invention consists in acomposition such as described above which comprises, moreover, as acombination product for simultaneous, separate or sequential use, ananti-tumoral antibody.

Most preferably, said second anti-tumoral antibody could be chosenthrough anti-IGF-IR, anti-EGFR, anti-HER2/neu, anti-VEGFR, anti-VEGF,etc., antibodies or any other anti-tumoral antibodies known by the manskilled in the art. It is evident that the use, as second antibody, offunctional fragments or derivatives of above mentioned antibodies ispart of the invention.

As a most preferred antibody, anti-EGFR antibodies are selected such asfor example the antibody C225 (Erbitux).

“Simultaneous use” is understood as meaning the administration of thetwo compounds of the composition according to the invention in a singleand identical pharmaceutical form.

“Separate use” is understood as meaning the administration, at the sametime, of the two compounds of the composition according to the inventionin distinct pharmaceutical forms.

“Sequential use” is understood as meaning the successive administrationof the two compounds of the composition according to the invention, eachin a distinct pharmaceutical form.

In a general fashion, the composition according to the inventionconsiderably increases the efficacy of the treatment of cancer. In otherwords, the therapeutic effect of the anti-c-Met antibodies according tothe invention is potentiated in an unexpected manner by theadministration of a cytotoxic agent. Another major subsequent advantageproduced by a composition according to the invention concerns thepossibility of using lower efficacious doses of active principle, whichallows the risks of appearance of secondary effects to be avoided or tobe reduced, in particular the effects of the cytotoxic agent.

In addition, this composition according to the invention would allow theexpected therapeutic effect to be attained more rapidly.

The composition of the invention can also be characterized in that itcomprises, moreover, as a combination product for simultaneous, separateor sequential use, a cytotoxic/cytostatic agent.

By “anti-cancer therapeutic agents” or “cytotoxic/cytostatic agents”, itis intended a substance which, when administered to a subject, treats orprevents the development of cancer in the subject's body. As nonlimitative example of such agents, it can be mentioned alkylatingagents, anti-metabolites, anti-tumor antibiotics, mitotic inhibitors,chromatin function inhibitors, anti-angiogenesis agents, anti-estrogens,anti-androgens or immunomodulators.

Such agents are, for example, cited in the 2001 edition of VIDAL, on thepage devoted to the compounds attached to the cancerology and hematologycolumn “Cytotoxics”, these cytotoxic compounds cited with reference tothis document are cited here as preferred cytotoxic agents.

More particularly, the following agents are preferred according to theinvention.

“Alkylating agent” refers to any substance which can cross-link oralkylate any molecule, preferably nucleic acid (e.g., DNA), within acell. Examples of alkylating agents include nitrogen mustard such asmechlorethamine, chlorambucol, melphalen, chlorydrate, pipobromen,prednimustin, disodic-phosphate or estramustine; oxazophorins such ascyclophosphamide, altretamine, trofosfamide, sulfofosfamide orifosfamide; aziridines or imine-ethylenes such as thiotepa,triethylenamine or altetramine; nitrosourea such as carmustine,streptozocin, fotemustin or lomustine; alkyle-sulfonates such asbusulfan, treosulfan or improsulfan; triazenes such as dacarbazine; orplatinum complexes such as cis-platinum, oxaliplatin and carboplatin.

“Anti-metabolites” refer to substances that block cell growth and/ormetabolism by interfering with certain activities, usually DNAsynthesis. Examples of anti-metabolites include methotrexate,5-fluoruracil, floxuridine, 5-fluorodeoxyuridine, capecitabine,cytarabine, fludarabine, cytosine arabinoside, 6-mercaptopurine (6-MP),6-thioguanine (6-TG), chlorodesoxyadenosine, 5-azacytidine, gemcitabine,cladribine, deoxycoformycin and pentostatin.

“Anti-tumor antibiotics” refer to compounds which may prevent or inhibitDNA, RNA and/or protein synthesis. Examples of anti-tumor antibioticsinclude doxorubicin, daunorubicin, idarubicin, valrubicin, mitoxantrone,dactinomycin, mithramycin, plicamycin, mitomycin C, bleomycin, andprocarbazine.

“Mitotic inhibitors” prevent normal progression of the cell cycle andmitosis. In general, microtubule inhibitors or taxoides such aspaclitaxel and docetaxel are capable of inhibiting mitosis. Vincaalkaloid such as vinblastine, vincristine, vindesine and vinorelbine arealso capable of inhibiting mitosis.

“Chromatin function inhibitors” or “topoisomerase inhibitors” refer tosubstances which inhibit the normal function of chromatin modelingproteins such as topoisomerase I or topoisomerase II. Examples ofchromatin function inhibitors include, for topoisomerase I,camptothecine and its derivatives such as topotecan or irinotecan, and,for topoisomerase II, etoposide, etoposide phosphate and teniposide.

“Anti-angiogenesis agent” refers to any drug, compound, substance oragent which inhibits growth of blood vessels. Exemplaryanti-angiogenesis agents include, but are by no means limited to,razoxin, marimastat, batimastat, prinomastat, tanomastat, ilomastat,CGS-27023A, halofuginon, COL-3, neovastat, BMS-275291, thalidomide, CDC501, DMXAA, L-651582, squalamine, endostatin, SU5416, SU6668,interferon-alpha,EMD121974, interleukin-12, IM862, angiostatin andvitaxin.

“Anti-estrogen” or “anti-estrogenic agent” refer to any substance whichreduces, antagonizes or inhibits the action of estrogen. Examples ofanti-estrogen agents are tamoxifen, toremifene, raloxifene, droloxifene,iodoxyfene, anastrozole, letrozole, and exemestane.

“Anti-androgens” or “anti-androgen agents” refer to any substance whichreduces, antagonizes or inhibits the action of an androgen. Examples ofanti-androgens are flutamide, nilutamide, bicalutamide, sprironolactone,cyproterone acetate, finasteride and cimitidine.

“Immunomodulators” are substances which stimulate the immune system.

Examples of immunomodulators include interferon, interleukin such asaldesleukine, OCT-43, denileukin diflitox and interleukin-2, tumoralnecrose factors such as tasonermine or others immunomodulators such aslentinan, sizofiran, roquinimex, pidotimod, pegademase, thymopentine,poly I:C or levamisole in conjunction with 5-fluorouracil.

For more detail, the man skill in the art could refer to the manualedited by the “Association Francaise des Enseignants de ChimieTherapeutique” and entitled “trait& de chimie therapeutique, vol. 6,Medicaments antitumoraux et perspectives dans le traitement des cancers,edition TEC & DOC, 2003”.

Can also be mentioned as chemical agents or cytotoxic agents, all kinaseinhibitors such as, for example, gefitinib or erlotinib.

In a particularly preferred embodiment, said composition as acombination product according to the invention is characterized in thatsaid cytotoxic agent is coupled chemically to said antibody forsimultaneous use.

In order to facilitate the coupling between said cytotoxic agent andsaid antibody according to the invention, it is especially possible tointroduce spacer molecules between the two compounds to be coupled, suchas poly(alkylene)glycols like polyethylene glycol, or else amino acids,or, in another embodiment, to use active derivatives of said cytotoxicagents into which would have been introduced functions capable ofreacting with said antibody according to the invention. These couplingtechniques are well known to the person skilled in the art and will notbe expanded upon in the present description.

The invention relates, in another aspect, to a composition characterizedin that one, at least, of said antibodies, or one of their functionalfragments or derivatives, is conjugated with a cell toxin and/or aradioelement.

Preferably, said toxin or said radioelement is capable of inhibiting atleast one cell activity of cells expressing the c-Met, in a morepreferred manner capable of preventing the growth or the proliferationof said cell, especially of totally inactivating said cell.

Preferably also, said toxin is an enterobacterial toxin, especiallyPseudomonas exotoxin A.

The radioelements (or radioisotopes) preferably conjugated to theantibodies employed for the therapy are radioisotopes which emit gammarays and preferably iodine¹³¹, yttrium⁹⁰, gold¹⁹⁹, palladium¹⁰⁰,copper⁶⁷, bismuth²¹⁷ and antimony²¹¹. The radioisotopes which emit betaand alpha rays can likewise be used for the therapy.

By toxin or radioelement conjugated to at least one antibody, or one ofits functional fragments, according to the invention, it is intended toindicate any means allowing said toxin or said radioelement to bind tosaid at least one antibody, especially by covalent coupling between thetwo compounds, with or without introduction of a linking molecule.

Among the agents allowing binding in a chemical (covalent),electrostatic or noncovalent manner of all or part of the components ofthe conjugate, mention may particularly be made of benzoquinone,carbodiimide and more particularly EDC(1-ethyl-3-[3-dimethyl-aminopropyl]-carbodiimide hydrochloride),dimaleimide, dithiobis-nitrobenzoic acid (DTNB), N-succinimidyl S-acetylthio-acetate (SATA), the bridging agents having one or more phenylazidegroups reacting with the ultraviolets (U.V.) and preferablyN-[-4-(azidosalicylamino)butyl]-3′-(2′-pyridyldithio)-propionamide(APDP), N-succinimid-yl 3-(2-pyridyldithio)propionate (SPDP),6-hydrazino-nicotinamide (HYNIC).

Another form of coupling, especially for the radioelements, can consistin the use of a bifunctional ion chelator.

Among these chelates, it is possible to mention the chelates derivedfrom EDTA (ethylenediaminetetraacetic acid) or from DTPA(diethylenetriaminepentaacetic acid) which have been developed forbinding metals, especially radioactive metals, and immunoglobulins.Thus, DTPA and its derivatives can be substituted by different groups onthe carbon chain in order to increase the stability and the rigidity ofthe ligand-metal complex (Krejcarek et al. (1977); Brechbiel et al.(1991); Gansow (1991); U.S. Pat. No. 4,831,175).

For example diethylenetriaminepentaacetic acid (DTPA) and itsderivatives, which have been widely used in medicine and in biology fora long time either in their free form, or in the form of a complex witha metallic ion, have the remarkable characteristic of forming stablechelates with metallic ions and of being coupled with proteins oftherapeutic or diagnostic interest such as antibodies for thedevelopment of radioimmunoconjugates in cancer therapy (Meases et al.,(1984); Gansow et al. (1990)).

Likewise preferably, said at least one antibody forming said conjugateaccording to the invention is chosen from its functional fragments,especially the fragments amputated of their Fc component such as thescFv fragments.

As already mentioned, in a preferred embodiment of the invention, saidcytotoxic/cytostatic agent or said toxin and/or a radioelement iscoupled chemically to at least one of the elements of said compositionfor simultaneous use.

The present invention comprises the described composition as amedicament.

The present invention moreover comprises the use of the compositionaccording to the invention for the preparation of a medicament.

In another aspect, the invention deals with the use of an antibody, orone of its functional fragments or derivatives, and/or of a compositionas above described for the preparation of a medicament intended toinhibit the growth and/or the proliferation of tumor cells.

Another aspect of the invention consists in the use of an antibody, orone of its functional fragments or derivatives and/or of a composition,as described above or the use above mentioned, for the preparation of amedicament intended for the prevention or for the treatment of cancer.

Is also comprises in the present invention a method intended to inhibitthe growth and/or the proliferation of tumor cells in a patientcomprising the administration to a patient in need thereof of anantibody, or one of its functional fragments or derivatives according tothe invention, an antibody produced by an hybridoma according to theinvention or a composition according to the invention.

The present invention further comprises a method for the prevention orthe treatment of cancer in a patient in need thereof, comprising theadministration to the patient of an antibody, or one of its functionalfragments or derivatives according to the invention, an antibodyproduced by an hybridoma according to the invention or a compositionaccording to the invention.

In a particular preferred aspect, said cancer is a cancer chosen fromprostate cancer, osteosarcomas, lung cancer, breast cancer, endometrialcancer, glioblastoma or colon cancer.

As explained before, an advantage of the invention is to allow thetreatment of HGF dependent and independent Met-activation relatedcancers.

The invention, in yet another aspect, encompasses a method of in vitrodiagnosis of illnesses induced by an overexpression or anunderexpression of the c-Met receptor starting from a biological samplein which the abnormal presence of c-Met receptor is suspected, saidmethod being characterized in that it comprises a step wherein saidbiological sample is contacted with an antibody of the invention, itbeing possible for said antibody to be, if necessary, labeled.

Preferably, said illnesses connected with an abnormal presence of c-Metreceptor in said diagnosis method will be cancers.

Said antibody, or one of its functional fragments, can be present in theform of an immunoconjugate or of a labeled antibody so as to obtain adetectable and/or quantifiable signal.

The antibodies labeled according to the invention or their functionalfragments include, for example, antibodies called immunoconjugates whichcan be conjugated, for example, with enzymes such as peroxidase,alkaline phosphatase, beta-D-galactosidase, glucose oxydase, glucoseamylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malatedehydrogenase or glucose 6-phosphate dehydrogenase or by a molecule suchas biotin, digoxygenin or 5-bromodeoxyuridine. Fluorescent labels can belikewise conjugated to the antibodies or to their functional fragmentsaccording to the invention and especially include fluorescein and itsderivatives, fluorochrome, rhodamine and its derivatives, GFP (GFP for“Green Fluorescent Protein”), dansyl, umbelliferone etc. In suchconjugates, the antibodies of the invention or their functionalfragments can be prepared by methods known to the person skilled in theart. They can be coupled to the enzymes or to the fluorescent labelsdirectly or by the intermediary of a spacer group or of a linking groupsuch as a polyaldehyde, like glutaraldehyde, ethylenediaminetetraaceticacid (EDTA), diethylene-triaminepentaacetic acid (DPTA), or in thepresence of coupling agents such as those mentioned above for thetherapeutic conjugates. The conjugates containing labels of fluoresceintype can be prepared by reaction with an isothiocyanate.

Other conjugates can likewise include chemoluminescent labels such asluminol and the dioxetanes, bio-luminescent labels such as luciferaseand luciferin, or else radioactive labels such as iodine¹²³, iodine¹²⁵,iodine¹²⁶, iodine¹³³, bromine⁷⁷, technetium^(99m), indiumm¹¹¹,indium^(113m), gallium⁶⁷, gallium⁶⁸, ruthenium⁹⁵, ruthenium⁹⁷,ruthenium¹⁰³, ruthenium¹⁰⁵, mercury¹⁰⁷, mercury²⁰³, rhenium^(99m),rhenium¹⁰¹, rhenium¹⁰⁵, scandium⁴⁷, tellurium^(121m), tellurium^(122m),tellurium^(125m), thulium¹⁶⁵, thulium¹⁶⁷, thulium¹⁶⁸, fluorine¹⁸,yttrium¹⁹⁹, iodine¹³¹. The methods known to the person skilled in theart existing for coupling the therapeutic radioisotopes to theantibodies either directly or via a chelating agent such as EDTA, DTPAmentioned above can be used for the radioelements which can be used indiagnosis. It is likewise possible to mention labeling with Na[I¹²⁵] bythe chloramine T method [Hunter W. M. and Greenwood F. C. (1962) Nature194:495] or else with technetium^(99m) by the technique of Crockford etal. (U.S. Pat. No. 4,424,200) or attached via DTPA as described byHnatowich (U.S. Pat. No. 4,479,930).

Thus, the antibodies, or their functional fragments, according to theinvention can be employed in a process for the detection and/or thequantification of an overexpression or of an underexpression, preferablyan overexpression, of the c-Met receptor in a biological sample,characterized in that it comprises the following steps:

a) the contacting of the biological sample with an antibody, or one ofits functional fragments, according to the invention; and

b) the demonstration of the c-Met/antibody complex possibly formed.

In a particular embodiment, the antibodies, or their functionalfragments, according to the invention, can be employed in a process forthe detection and/or the quantification of the c-Met receptor in abiological sample, for the monitoring of the efficacy of a prophylacticand/or therapeutic treatment of c-Met-dependent cancer.

More generally, the antibodies, or their functional fragments, accordingto the invention can be advantageously employed in any situation wherethe expression of the c-Met-receptor must be observed in a qualitativeand/or quantitative manner.

Preferably, the biological sample is formed by a biological fluid, suchas serum, whole blood, cells, a tissue sample or biopsies of humanorigin.

Any procedure or conventional test can be employed in order to carry outsuch a detection and/or dosage. Said test can be a competition orsandwich test, or any test known to the person skilled in the artdependent on the formation of an immune complex of antibody-antigentype. Following the applications according to the invention, theantibody or one of its functional fragments can be immobilized orlabeled. This immobilization can be carried out on numerous supportsknown to the person skilled in the art. These supports can especiallyinclude glass, polystyrene, poly-propylene, polyethylene, dextran,nylon, or natural or modified cells. These supports can be eithersoluble or insoluble.

By way of example, a preferred method brings into play immunoenzymaticprocesses according to the ELISA technique, by immunofluorescence, orradio-immunoassay (RIA) technique or equivalent.

Thus, the present invention likewise comprises the kits or setsnecessary for carrying out a method of diagnosis of illnesses induced byan overexpression or an underexpression of the c-Met receptor or forcarrying out a process for the detection and/or the quantification of anoverexpression or of an underexpression of the c-Met receptor in abiological sample, preferably an overexpression of said receptor,characterized in that said kit or set comprises the following elements:

a) an antibody, or one of its functional fragments, according to theinvention;

b) optionally, the reagents for the formation of the medium favorable tothe immunological reaction;

c) optionally, the reagents allowing the demonstration of c-Met/antibodycomplexes produced by the immunological reaction.

A subject of the invention is likewise the use of an antibody or acomposition according to the invention for the preparation of amedicament intended for the specific targeting of a biologically activecompound to cells expressing or overexpressing the c-Met receptor.

It is intended here by biologically active compound to indicate anycompound capable of modulating, especially of inhibiting, cell activity,in particular their growth, their proliferation, transcription or genetranslation.

A subject of the invention is also an in vivo diagnostic reagentcomprising an antibody according to the invention, or one of itsfunctional fragments, preferably labeled, especially radiolabeled, andits use in medical imaging, in particular for the detection of cancerconnected with the expression or the overexpression by a cell of thec-Met receptor.

The invention likewise relates to a composition as a combination productor to an anti-c-Met/toxin conjugate or radioelement, according to theinvention, as a medicament.

Preferably, said composition as a combination product or said conjugateaccording to the invention will be mixed with an excipient and/or apharmaceutically acceptable vehicle.

In the present description, pharmaceutically acceptable vehicle isintended to indicate a compound or a combination of compounds enteringinto a pharmaceutical composition not provoking secondary reactions andwhich allows, for example, facilitation of the administration of theactive compound(s), an increase in its lifespan and/or in its efficacyin the body, an increase in its solubility in solution or else animprovement in its conservation. These pharmaceutically acceptablevehicles are well known and will be adapted by the person skilled in theart as a function of the nature and of the mode of administration of theactive compound(s) chosen.

Preferably, these compounds will be administered by the systemic route,in particular by the intravenous route, by the intramuscular,intradermal, intraperitoneal or subcutaneous route, or by the oralroute. In a more preferred manner, the composition comprising theantibodies according to the invention will be administered severaltimes, in a sequential manner.

Their modes of administration, dosages and optimum pharmaceutical formscan be determined according to the criteria generally taken into accountin the establishment of a treatment adapted to a patient such as, forexample, the age or the body weight of the patient, the seriousness ofhis/her general condition, the tolerance to the treatment and thesecondary effects noted.

Other characteristics and advantages of the invention appear in thecontinuation of the description with the examples and the figureswherein:

FIG. 1: Examples of FACS profiles of the selected anti-c-Met antibodies;

FIGS. 2 A and 2B: In vitro inhibition of BXPC3 proliferation byantibodies targeting c-Met;

FIG. 3: Inhibition of c-Met dimerization;

FIG. 4: Protein recognition by anti-c-Met antibodies;

FIGS. 5A and 5B: “Epitope mapping” of 11E1 and 5D5 by BIAcore analysis;

FIGS. 6A and 6B: Effect of MAbs on c-Met phosphorylation;

FIGS. 7A and 7B: Displacement of radio-labeled HGF by anti-c-Metantibodies;

FIG. 8: Inhibition of invasion by anti-c-Met antibodies [in this figure,SVF means Fetal Calf Serum (FCS)];

FIG. 9: Effect of anti c-Met antibodies on wound healing;

FIGS. 10A and 10B: Scatter assay;

FIG. 11: Three-dimensional tubulogenesis assay;

FIGS. 12A and 12B: Effect of antibodies on spheroid formation;

FIG. 13: In vivo activity of anti-c-Met Mabs in the U87MG xenograftmodel;

FIG. 14: HGF expression by a set of tumour cell lines;

FIGS. 15A and 15B: Characterization of the NCI-H441 cell line; with FIG.15A corresponding to quantitative RT-PCR analysis and FIG. 15Bcorresponding to FACS analysis;

FIG. 16: In vivo activity of anti-c-Met antibodies on NCI-H441 xenograftmodel;

FIG. 17A: Alignment of 224G11 VL to murine IGKV3-5*01 germline gene;

FIG. 17B: Alignment of 224G11 VL to murine IGKJ4*01 germline gene;

FIG. 18A: Alignment of 224G11 VL to human IGKV3-11*01 and IGKV4-1*01germline genes;

FIG. 18B: Alignment of 224G11 VL to human IGKJ4*02 germline gene;

FIG. 19A: IGKV3-11*01 based humanized version of 224G11 VL withmentioned mutations;

FIG. 19B: IGKV4-1*01 based humanized version of 224G11 VL with mentionedmutations;

FIG. 20A: Alignment of 224G11 VH to murine IGHV1-18*01 germline gene;

FIG. 20B: Alignment of 224G11 VH to murine IGHD2-4*01 germline gene;

FIG. 20C: Alignment of 224G11 VH to murine IGHJ2*01 germline gene;

FIG. 21A: Alignment of 224G11 VH to human IGHV1-2*02 germline gene;

FIG. 21B: Alignment of 224G11 VH to human IGHJ4*01 germline gene;

FIG. 22: Humanized 224G11 VH with mentioned mutations;

FIG. 23A: Alignment of 227H1 VL to murine IGKV3-5*01 germline gene;

FIG. 23B: Alignment of 227H1 VL to murine IGKJ4*01 germline gene;

FIG. 24A: Alignment of 227H1 VL to human IGKV3-11*01 and IGKV4-1*01germline genes;

FIG. 24B: Alignment of 227H1 VL to human IGKJ4*02 germline gene;

FIG. 25A: IGKV3-11*01 based humanized version of 227H1 VL with mentionedmutations;

FIG. 25B: IGKV4-1*01 based humanized version of 227H1 VL with mentionedmutations;

FIG. 26A: Alignment of 227H1 VH to murine IGHV1-18*01 germline gene;

FIG. 26B: Alignment of 227H1 VH to murine IGHD1-1*02 germline gene;

FIG. 26C: Alignment of 227H1 VH to murine IGHJ2*01 germline gene;

FIG. 27A: Alignment of 227H1 VH to human IGHV1-2*02 germline gene;

FIG. 27B: Alignment of 227H1 VH to human IGHJ4*01 germline gene;

FIG. 28: Humanized 227H1 VH with mentioned mutations;

FIG. 29A: Alignment of 223C4 VL to murine IGKV12-46*01 germline gene;

FIG. 29B: Alignment of 223C4 VL to murine IGKJ2*01 germline gene;

FIG. 30A: Alignment of 223C4 VL to human IGKV1-NL1*01 germline gene;

FIG. 30B: Alignement of 223C4 VL to human IGKJ2*01 germline gene;

FIG. 31: Humanized 223C4 VL with mentioned mutations;

FIG. 32A: Alignment of 223C4 VH to murine IGHV1-18*01 germline gene;

FIG. 32B: Alignment of 223C4 VH to murine IGHD6-3*01 germline gene;

FIG. 32C: Alignment of 223C4 VH to murine IGHJ4*01 germline gene;

FIG. 33A: Alignment of 223C4 VH to human IGHV1-2*02 germline gene;

FIG. 33B: Alignment of 223C4 VH to human IGHD1-26*01 germline gene;

FIG. 33C: Alignment of 223C4 VH to human IGHJ6*01 germline gene; and

FIG. 34: Humanized 223C4 VH with mentioned mutations;

FIG. 35: Anti-tumor activity of the murine 224G11 Mab alone or combinedwith Navelbine® on the established xenograft NCI-H441 tumor model;

FIG. 36: Evaluation of anti-c-Met Mabs on HUVEC proliferation;

FIG. 37: Evaluation of anti-c-Met Mabs on HUVEC tube formation;

FIG. 38A: Alignment of 11E1 VL to murine IGKV4-79*01 germline gene;

FIG. 38B: Alignment of 11E1 VL to murine IGKJ4*01 germline gene;

FIG. 39A: Alignment of 11E1 VL to human IGKV3D-7*01 germline gene;

FIG. 39B: Alignment of 11E1 VL to human IGKJ4*02 germline gene;

FIG. 40: Humanized version of 11E1 VL with mentioned mutations;

FIG. 41A: Alignment of 11E1 VH to murine IGHV1-7*01 germline gene;

FIG. 41B: Alignment of 11E1 VH to murine IGHD4-1*01 germline gene;

FIG. 41C: Alignment of 11E1 VH to murine IGHJ3*01 germline gene;

FIG. 42A: Alignment of 11E1 VH to human IGHV1-2*02 and IGHV1-46*01germline genes;

FIG. 42B: Alignment of 11E1 VH to human IGHJ4*03 germline gene;

FIG. 43: Humanized 11E1 VH with mentioned mutations;

FIGS. 44A and 44B: c-Met Phosphorylation assay on A549 cells. Evaluationof 11E1 and 224G11 purified Mabs, in absence or in presence of HGF,either at 30 μg/ml (FIG. 44A) or within a dose range from 0.0015 to 30μg/ml in order to determine EC₅₀ values (FIG. 44B);

FIG. 45: In vivo combination of 224G11 Mab with Navelbine® in the NSCLCNCI-H441 xenograft model;

FIG. 46: In vivo combination of 224G11 Mab with Doxorubicin in the NSCLCNCI-H441 xenograft model;

FIG. 47: In vivo combination of 224G11 Mab with Docetaxel in the NSCLCNCI-H441 xenograft model;

FIG. 48: In vivo combination of 224G11 Mab with Temozolomide in theNSCLC NCI-H441 xenograft model;

FIGS. 49A, 49B, 49C and 49D: Effect of anti-c-Met Mabs on U87-MGspheroid growth;

FIGS. 50A and 50B: In vitro activity of chimeric and humanized forms of224G11 in the phospho-c-Met assay;

FIG. 51: Settings of Biacore analysis;

FIG. 52: In vivo activity of 224G11 on MDA-MB-231 cells co-implantedwith MRCS cells as human HGF source on Athymic nude mice;

FIG. 53: ELISA based binding assay to Fc-cMet. Anti-Fc-c-Met bindingactivity was measured in an ELISA-based assay where anti-murine Fcconjugates was used to detect the purified murine monoclonal antibodies11E1, 224G11 and 227H1. Dose-dependent binding activities ontoplastic-coated recombinant Fc-cMet was measured at 450 nm;

FIG. 54: HGF-cMet competition assay. In this ELISA-based assay,recombinant Fc-cMET residual binding to plastic coated HGF in thepresence of purified murine monoclonal antibodies 11E1, 224G11 and 227H1was detected with anti-murine Fc conjugate and measured at 450 nm;

FIG. 55: Amino acid sequences alignment of 227H1-derived recombinant VHdomains. The 227H1 VH amino acid sequence is aligned with the selectedhuman receiving framework sequence, with only mentioned the amino acidsthat were found different from the murine 227H1 VH sequence. 227H1 HZ1,HZ2 and HZ3 VH sequences correspond to implemented humanized versions ofthe 227H1 murine VH domain, with remaining murine residues in bold. InHZ3, 10 residues (*) were automatically changed for their humancounterparts. In HZ2, the seven residues from the third group (3) havebeen studied. In HZ1VH, the nine residues from the second group (2) havebeen mutated into their human counterparts, only the six residues fromthe first group (1) remain murine;

FIG. 56: ELISA based binding assay to Fc-cMet of recombinant 227H1antibodies. Anti-Fc-cMet binding activity was measured in an ELISA-basedassay where anti-human Fc conjugates was used to detect chimeric andhumanized 227H1-derived recombinant antibodies. Dose-dependent bindingactivities onto plastic-coated recombinant Fc-cMet of humanized VHdomains-derived 227H1 antibodies was measured at 450 nm and then compareto those of the parental/reference chimeric antibody;

FIG. 57: ELISA based binding assay to Fc-cMet of recombinant227H1-derived antibodies. Anti-Fc-cMet binding activity was measured inan ELISA-based assay where anti-human Fc conjugates was used to detectchimeric and humanized 227H1-derived recombinant antibodies.Dose-dependent binding activity onto plastic-coated recombinant Fc-cMetof humanized HZ4VH-derived 227H1 antibody was measured at 450 nm andthen compared to those of the parental/reference chimeric antibody;

FIG. 58: HGF-cMet competition assay of 227H1 murine and recombinantantibodies. In this ELISA-based assay, recombinant Fc-cMet residualbinding to plastic coated HGF in the presence of the different forms ofthe 227H1 antibody was detected with a biotinylated unrelated anti-cMetantibody. Purified murine 227H1 monoclonal antibody, chimeric andHZ4VH-derived humanized 227H1-derived recombinant antibodies were testedand compared for their abilities to compete with HGF-cMet binding whenmeasured at 450 nm;

FIG. 59: 227H1-HZ VH humanized variable domain sequence. *, correspondsto amino acids changed de facto to their human counterparts; !,corresponds to amino acids humanized during the HZ3 to HZ1implementation; §, corresponds to amino acids humanized in the final227H1-HZ VH sequence;

FIG. 60: Amino acid sequences alignment of 11E1-derived recombinant VHdomains. The 11E1 VH amino acid sequence is aligned with the selectedhuman receiving framework sequence, with only mentioned the amino acidsthat were found different from the murine 11E1 VH sequence. 11E1 HZ VH1,VH2 and VH3 sequences correspond to implemented humanized versions ofthe 11E1 murine VH domain, with remaining murine residues in bold. In HZVH3, seven residues (*) were automatically changed for their humancounterparts. In HZ VH2, the seven residues from the third group (3)have been studied. In HZ VH1, the five residues from the second group(2) have been mutated into their human counterparts, only the fiveresidues from the first group (1) remain murine;

FIG. 61: ELISA based binding assay to Fc-cMet of recombinant 11E1antibodies. Anti-Fc-cMet binding activity was measured in an ELISA-basedassay where anti-human Fc conjugates was used to detect chimeric andhumanized 11E1-derived recombinant antibodies. Dose-dependent bindingactivities onto plastic-coated recombinant Fc-cMet of humanized VHdomains-derived 11E1 antibodies was measured at 450 nm and then compareto those of the parental/reference chimeric antibody;

FIG. 62: Amino acid sequences alignment of 11E1-derived recombinant VLdomains. The 11E1 VL amino acid sequence is aligned with the selectedhuman receiving framework sequence, with only mentioned the amino acidsthat were found different from the murine 11E1 VL sequence. 11E1 HZ VL1,VL2 and VL3 sequences correspond to implemented humanized versions ofthe 11E1 murine VL domain, with remaining murine residues in bold. In HZVL3, ten residues (*) were automatically changed for their humancounterparts. In HZ VL2, the eight residues from the third group (3)have been studied. In HZ VL1, the eight residues from the second group(2) have been mutated into their human counterparts, only the fourresidues from the first group (1) remain murine;

FIG. 63: ELISA based binding assay to Fc-cMet of recombinant 11E1antibodies. Anti-Fc-cMet binding activity was measured in an ELISA-basedassay where anti-human Fc conjugates was used to detect chimeric andhumanized 11E1-derived recombinant antibodies. Dose-dependent bindingactivities onto plastic-coated recombinant Fc-cMet of humanized VLdomains-derived 11E1 antibodies was measured at 450 nm and then compareto those of the parental/reference chimeric antibody;

FIG. 64: ELISA based binding assay to Fc-cMet of recombinant 11E1antibodies. Anti-Fc-cMet binding activity was measured in an ELISA-basedassay where anti-human Fc conjugates was used to detect chimeric andhumanized 11E1-derived recombinant antibodies. Dose-dependent bindingactivities onto plastic-coated recombinant Fc-cMet of single or doublehumanized domains-derived 11E1 antibodies was measured at 450 nm andthen compared to those of the parental/reference chimeric antibody;

FIG. 65: Amino acid sequences alignment of 224G11 VH domain sequence.The 224G11 VH amino acid sequence is aligned with the 227H1 VH sequence(underlined are non homologous residues) and with the selected humanreceiving framework sequence, with only mentioned the amino acids thatwere found different from the murine 224G11 VH sequence. 224G11 HZ VH0sequence correspond to “227H1-based/full-IMGT” humanized version of the224G11 murine VH domain. In this sequence no outside-IMGT-CDRs residuesremain murine;

FIG. 66: ELISA based binding assay to Fc-cMet of recombinant 224G11antibodies. Anti-Fc-cMet binding activity was measured in an ELISA-basedassay where anti-human Fc conjugates was used to detect chimeric andHZVHO-derived humanized 224G11-derived recombinant antibodies.Dose-dependent binding activity onto plastic-coated recombinant Fc-cMetof the HZVHO “full-IMGT” humanized VH domain-derived 224G11 antibody wasmeasured at 450 nm and then compared to those of the parental/referencechimeric antibody;

FIG. 67: HGF-cMet competition assay of 224G11 murine and recombinantantibodies. In this ELISA-based assay, recombinant Fc-cMet residualbinding to plastic coated HGF in the presence of the different forms ofthe 224G11 antibody was detected with a biotinylated unrelated anti-cMetantibody. Purified murine 224G11 monoclonal antibody, chimeric andHZVHO-derived humanized 224G11-derived recombinant antibodies weretested and compared for their abilities to compete with HGF-cMet bindingwhen measured at 450 nm;

FIG. 68: Amino acid sequences alignment of 224G11 VL domain sequences.The 224G11 VL amino acid sequence is aligned with the two selected humanreceiving framework sequences, with only mentioned the amino acids thatwere found different from the murine 224G11 VL sequence. 224G11 HZ VL3sequence correspond to “shorter-CDR1” humanized version of the 224G11murine VH domain while HZ VL6 correspond to the “longer-CDR1” version,with the remaining murine residues in bold. For both basic humanizedversions, the remaining murine residues are ranked for furtherhumanization process where * corresponds to amino acids humanized in thebasic versions, and 3, 2 and 1 correspond to the residues groups for thedesign of the implemented humanized versions;

FIG. 69: ELISA based binding assay to Fc-cMet of recombinant 224G11antibodies. Anti-Fc-cMet binding activity was measured in an ELISA-basedassay where anti-human Fc conjugates was used to detect chimeric andhumanized 22G11-derived recombinant antibodies. Dose-dependent bindingactivities onto plastic-coated recombinant Fc-cMet of humanized VL3 andVL6 domains-derived 224G11 antibodies was measured at 450 nm and thencompare to those of the parental/reference chimeric antibody;

FIG. 70: ELISA based binding assay to Fc-cMet of recombinant 224G11antibodies. Anti-Fc-cMet binding activity was measured in an ELISA-basedassay where anti-human Fc conjugates was used to detect chimeric andhumanized 224G11-derived recombinant antibodies. Dose-dependent bindingactivities onto plastic-coated recombinant Fc-cMet of humanized VLdomains-derived 224G11 antibodies was measured at 450 nm and thencompare to those of the parental/reference chimeric antibody;

FIG. 71: HGF-cMet competition assay of 224G11 murine and recombinantantibodies. In this ELISA-based assay, recombinant Fc-cMet residualbinding to plastic coated HGF in the presence of the different forms ofthe 224G11 antibody was detected with a biotinylated unrelated anti-cMetantibody. Purified murine 224G11 monoclonal antibody, chimeric and HZVL4-derived humanized 224G11-derived recombinant antibodies were testedand compared for their abilities to compete with HGF-cMet binding whenmeasured at 450 nm;

FIG. 72: Amino acid sequence of VL4 humanized 224G11 VL domain sequence.*, corresponds to amino acids changed de facto to their humancounterparts in the basic HZ VL6 version; !, corresponds to amino acidshumanized during the HZ VL6 to HZ VL4 implementation; §, corresponds toamino acids that remain murine in the 224G11-HZ VL4 sequence;

FIG. 73: ELISA based binding assay to Fc-cMet of recombinant 224G11antibodies. Anti-Fc-cMet binding activity was measured in an ELISA-basedassay where anti-human Fc conjugates was used to detect chimeric andhumanized 22G11-derived recombinant antibodies. Dose-dependent bindingactivities onto plastic-coated recombinant Fc-cMet of single- ordouble-humanized domains-derived 224G11 antibodies was measured at 450nm and then compare to those of the parental/reference chimericantibody;

FIG. 74: HGF-cMet competition assay of 224G11 murine and recombinantantibodies. In this ELISA-based assay, recombinant Fc-cMet residualbinding to plastic coated HGF in the presence of the different forms ofthe 224G11 antibody was detected with a biotinylated unrelated anti-cMetantibody. Purified murine 224G 11 monoclonal antibody, chimeric andfully humanized 224G11-derived recombinant antibodies were tested andcompared for their abilities to compete with HGF-cMet binding whenmeasured at 450 nm;

FIG. 75: ELISA based binding assay to Fc-cMet of recombinant 224G11antibodies. Anti-Fc-cMet binding activity was measured in an ELISA-basedassay where anti-human Fc conjugates was used to detect chimeric andhumanized 22G11-derived recombinant antibodies. Dose-dependent bindingactivities onto plastic-coated recombinant Fc-cMet of single mutants ofthe VL4-derived fully humanized 224G11 antibodies was measured at 450 nmand then compare to those of the parental/reference chimeric antibody;

FIG. 76: ELISA based binding assay to Fc-cMet of recombinant 224G11antibodies. Anti-Fc-cMet binding activity was measured in an ELISA-basedassay where anti-human Fc conjugates was used to detect chimeric andhumanized 22G11-derived recombinant antibodies. Dose-dependent bindingactivities onto plastic-coated recombinant Fc-cMet of single andmultiple mutants of the VL4-derived fully humanized 224G11 antibodieswas measured at 450 nm and then compare to those of theparental/reference chimeric antibody; and

FIG. 77: HGF-cMet competition assay of 224G11 murine and recombinantantibodies. In this ELISA-based assay, recombinant Fc-cMet residualbinding to plastic coated HGF in the presence of the different forms ofthe 224G11 antibody was detected with a biotinylated unrelated anti-cMetantibody. Purified murine 224G11 monoclonal antibody, chimeric andsingle or multiple mutants of the VL4-derived fully humanized 224G11recombinant antibodies were tested and compared for their abilities tocompete with HGF-cMet binding when measured at 450 nm.

EXAMPLE 1 Generation of Antibodies Against c-Met

To generate anti-c-Met antibodies 8 weeks old BALB/c mice were immunizedeither 3 to 5 times subcutaneously with a CHO transfected cell line thatexpress c-Met on its plasma membrane (20×10⁶ cells/dose/mouse) or 2 to 3times with a c-Met extracellular domain fusion protein (10-15μg/dose/mouse) (R&D Systems, Catalog #358MT) or fragments of thisrecombinant protein mixed with complete Freund adjuvant for the firstimmunization and incomplete Freund adjuvant for the following ones.Mixed protocols in which mice received both CHO-cMet cells andrecombinant proteins were also performed. Three days before cell fusion,mice were boosted i.p. or i.v. with the recombinant protein orfragments. Then spleens of mice were collected and fused to SP2/0-Ag14myeloma cells (ATCC) and subjected to HAT selection. Four fusions wereperformed. In general, for the preparation of monoclonal antibodies ortheir functional fragments, especially of murine origin, it is possibleto refer to techniques which are described in particular in the manual“Antibodies” (Harlow and Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor N.Y., pp. 726, 1988) or tothe technique of preparation of hybridomas described by Kohler andMilstein (Nature, 256:495-497, 1975).

Obtained hybridomas were initially screened by ELISA on the c-Metrecombinant protein and then by FACS analysis on A549 NSCLC, BxPC3pancreatic, and U87-MG glioblastoma cell lines (representative profileswere presented in FIG. 1) to be sure that the produced antibodies willbe able to also recognize the native receptor on tumor cells. Positivereactors on these 2 tests were amplified, cloned and a set of hybridomaswas recovered, purified and screened for its ability to inhibit in vitrocell proliferation in the BxPC3 model.

For that purpose 50 000 BxPC3 cells were plated in 96 well plates inRPMI medium, 2 mM L. Glutamine, without SVF. 24 hours after plating,antibodies to be tested were added at a final concentration ranging from0.0097 to 40 ng/ml 60 min before addition of 100 ng/ml of hHGF. After 3days, cells were pulsed with 0.5 nCi of [³H]thymidine for 16 hours. Themagnitude of [³H]thymidine incorporated into trichloroaceticacid-insoluble DNA was quantified by liquid scintillation counting.Results were expressed as raw data to really evaluate the intrinsicagonistic effect of each Mab (FIGS. 2A and 2B).

Then antibodies inhibiting at least 50% cell proliferation wereevaluated as supernatants by BRET analysis on c-Met transfected cells.For that purpose, CHO stable cell lines expressing C-Met-Rluc orC-Met-Rluc and C-Met-K1100A-YFP were generated. Cells were distributedin white 96 well microplates in DMEM-F12/FBS 5% culture medium one ortwo days before BRET experiments. Cells were first cultured at 37° C.with CO2 5% in order to allow cell attachment to the plate. Cells werethen starved with 200 μl DMEM/well overnight Immediately prior to theexperiment, DMEM was removed and cells quickly washed with PBS. Cellswere incubated in PBS in the presence or absence of antibodies to betested or reference compounds, 10 min at 37° C. prior to the addition ofcoelenterazine with or without HGF in a final volume of 50 μl. Afterincubation for further 10 minutes at 37° C., light-emission acquisitionat 485 nm and 530 nm was initiated using the Mithras luminometer(Berthold) (1 s/wave length/well repeated 15 times).

BRET ratio has been defined previously [Angers et al., Proc. Natl. Acad.Sci. USA, 2000, 97:3684-3689] as: [(emission at 530 nm)−(emission at 485nm)×Cf]/(emission at 485 nm), where Cf corresponds to (emission at 530nm)/(emission at 485 nm) for cells expressing Rluc fusion protein alonein the same experimental conditions. Simplifying this equation showsthat BRET ratio corresponds to the ratio 530/485 nm obtained when thetwo partners were present, corrected by the ratio 530/485 nm obtainedunder the same experimental conditions, when only the partner fused toR. reniformis luciferase was present in the assay. For the sake ofreadability, results are expressed in milliBRET units (mBU); mBUcorresponds to the BRET ratio multiplied by 1000.

After this second in vitro test, 4 antibodies i) without intrinsicactivity as a whole molecule in the functional test of proliferation,ii) inhibiting significantly BxPC3 proliferation (FIGS. 2A and 2B) andiii) inhibiting c-Met dimerization (FIG. 3) were selected. These 3antibodies of IgG1 kappa isotype were described as 11E1, 224G11, 223C4and 227H1. In the experiments, the 5D5 Mab, generated by Genentech, andavailable at the ATCC, was added as a control for the intrinsicagonistic activity.

FIGS. 2A and 2B demonstrates that 11E1, 224G11, 223C4 and 227H1 werewithout any agonist activity in contrast to 5D5 which induced a dosedependent stimulation of cell proliferation in absence of ligand. Asignificant inhibition of cell proliferation was observed with the 4selected antibodies. 5D5 is without effect on HGF-induced cellproliferation in this test.

When evaluated for blockade of dimerization significant effects reaching32, 55, 69 and 52% inhibition of dimerization for 224G11, 223C4,11E1 and227H1 respectively were observed. Compared to basal signals in therespective experiments, 5D5 antibody is without effect in thisdimerization model.

EXAMPLE 2 Protein Recognition by Anti-c-Met Antibodies

To characterize the pattern of recognition of the 3 selected antibodies,3 ELISA have been set up with the recombinant c-Met protein, itsmonomeric fragment (obtained by cleavage of the recombinant c-Met-Fcprotein and the recombinant SEMA domain.

Results presented in FIG. 4 demonstrated that the 4 antibodiesrecognized both dimeric and monomeric proteins. To perform these ELISAthe human dimeric c-Met protein (R&D sytems, cat#358MT) is coated at theconcentration of 0.7 μg/ml in PBS overnight at 4° C. After saturation ofthe plates (Costar #3690) with a 0.5% gelatin solution 2 hours at 37°C., hybridoma supernatants are incubated 1 hour at 37° C. Once rinsedwith PBS, the anti-mouse HRP-antibody (Jackson ImmunoResearch, catalog#115-035-164) is added to each well at a 1/5000 dilution in ELISA buffer(0.1% gelatin/0.05% Tween 20 in PBS) and the plates incubated for 1 hourat 37° C. After 3 washes in PBS, the activity of the peroxydase isrevealed by the addition of 50 μl of TMB substrate (Uptima). Thereaction is left to occur for 5 min at room temperature. The reaction isstopped by the addition of 50 μl/well of a 1 M H₂SO₄ solution and readon a plate reader at 450 nm. The same kind of protocol was performed onmonomeric c-Met and SEMA domain but in that cases proteins were coatedat 5 and 3 μg/ml respectively.

The 5D5 Mab introduced as a positive control recognized as expected theSEMA protein. 224G11, 227H1 and 223C4 did not bind the SEMA domain. 11E1is able to bind the SEMA.

To determine whether 11E1 and 5D5, both recognizing the SEMA domaincompete for overlapping epitopes, BIAcore analysis were performed.BIAcore system based on the Surface Plasmon Resonance phenomenon deliverdata by monitoring binding events in real-time. It is then useful togroup antibodies in a so called “epitope mapping” experiments. A coupleof antibodies unable to bind at the same time on the antigen moleculeare classified in the same group (identical or neighbouring bindingsites). At the opposite when their respective binding sites aresufficiently distant to allow a simultaneous binding of both antibodiesthese later are classified into two different groups. In suchexperiments, the antigen is commonly used as the ligand (immobilized onthe sensorchip) and the antibodies are used without any labelling asanalytes (solution phase).

All the experiments described have been done on a BIAcore X instrument(GE Healthcare Europe GmbH). A CMS sensorchip (BIAcore) activated by amouse anti-Tag-6H is Mab (R&D System ref MAB050) has been preparedfollowing the manufacturer instructions by using the amine coupling kit(BIAcore). The running buffer (HBS-EP) and regeneration buffer (Glycine,HCl) are from BIAcore. A recombinant soluble version of the human HGFreceptor produced as a chimeric molecule c-Met-Fc-Tag H is was from R&Dsystems (ref 358-MT-CF). The experiments were done at 25° C., at a flowrate of 30 nl/min. A 10 ng/ml solution of c-Met in running buffer wasinjected during one minute on the flowcell2 (fc2) typically 270 RU ofthe soluble form of c-Met were captured. The flowcell1 (fc1) was used asa reference to check any non specific binding of the antibodies to thesensorchip matrix.

Sequential injections of antibodies to be tested were performed. Anantibody was injected on both flowcells during 2 minutes. A secondantibody (or the same) was then injected in the same conditions. If nosignificant binding was observed a third injection was done with anotherantibody. The sensorchip was then regenerated by a single 30 s injectionof the regeneration buffer. Either antibodies and c-Met-Fc werediscarded at this stage.

Analysis of the Results:

The ability of an antibody “A” to block the binding of an antibody “B”is calculated by the ratio BIA/C=(R2A/B/R1B)×100: where R2A/B is theresponse corresponding to the binding of the MAb “B” when it wasinjected after Mab “A” and R1B is the response corresponding to thebinding of the MAb “B” when it was injected first. A BIA/C below 20%means that A is able to block the binding of B so that A and B haveneighbouring binding sites.

The epitope mapping has been performed with 2 Mabs, 11E1 and 5D5.

TABLE 3 2^(nd) Ab (B) 1^(st) Ab (A) 11E1 5D5 11E1 6.5% 84.2% 5D5 98.4%11.0%

Visualisation of the binding on around 270RU of captured c-Met-Fc by thesequential 2 minutes injections of Mabs 5D5 (first), 5D5 (second) and11E1 (third) at a concentration of 10 ng/ml each demonstrated that 5D5and 11E1 bind clearly to two distant sites (FIG. 5A). This observationwas confirmed by the reciprocal sequence of antibody (FIG. 5B).

Table 3 summarized the calculation ratio obtained with the differentsequences of these 2 antibodies. Black values (over 75%) mean that Mab Adoes not block the binding of Mab B. Bold/italic values (below 20%) meanthat the binding sites of both antibody (A and B) are identical orsufficiently close to unable a simultaneous binding.

EXAMPLE 3 Effect of Mabs on c-Met Phosphorylation

To determine the activity of anti-c-Met antibodies on c-Metphosphorylation a phospho c-Met ELISA assay was set-up. Briefly 500 000A549 cells were seeded in each well of 6-well plates in F12K medium+10%FCS. 16 hours before HGF addition (100 ng/ml), cells were starved andeach antibody to be tested was added at a final concentration of 30ng/ml 15 minutes before ligand stimulation. 15 minutes after HGFaddition, cold lysis buffer was added, cells were scraped and celllysates collected and centrifuged at 13 000 rpm for 10 min at 4° C.Supernatants were quantified with a BCA kit (Pierce) and stored at −20°C. For ELISA assay, a goat anti-c-Met antibody (R&D ref. AF276) was usedas a capture antibody (coating overnight at 4° C.) and after asaturation step (1 h at RT) with a TBS-BSA 5% buffer, 25 ng of proteinfrom the different cell lysates was added to each well of the 96-wellplate. After a 90 minute-incubation time at RT, plates were washed fourtimes and an anti-phospho-c-Met antibody (Rabbit anti-pY1230-1234-1235c-Met) was added. After an additional 1 hour incubation time and 4washes an anti-rabbit-HRP (Biosource) was added for 1 hour at RT andthen Luminol substrate was added before evaluation the luminescence witha Mithras device. Results presented in FIG. 6B demonstrated that 11E1,224G11, 223C4 and 227H1 inhibit c-Met phosphorylation by 68, 54, 80 and65% respectively compared to the 5D5 Mab which displayed a weakerinhibition of c-Met phosphorylation (42%). In this test, a weak basaleffect (less to 20%) was observed with the 4 candidate antibodies (FIG.6A). As described in the various examples presented in this patent, thisweak basal effect has no consequences on the activity of antibodies inother in vitro and in vivo tests. The 5D5 used as a control displayed,in this test a significant basal effect.

EXAMPLE 4 Displacement of Radio-Labelled HGF by Anti-c-Met Antibodies

To determine whether the anti-c-Met antibodies were able to displaceHGF, binding experiments were set up. Briefly, protein A FlashPlate96-well microplates (Perkin Elmer) were saturated with 0.5% gelatine inPBS (200 μl/well, 2 h at room temperature) before adding recombinantc-Met-Fc (R&D Systems) as a coating protein. Two thousand μl of a 1μg/ml c-Met-Fc solution in PBS were added to each well. Plates were thenincubated overnight at 4° C. Free residual Protein A sites were furthersaturated with a non relevant hIgG (0.5 μg/well in PBS) for 2 h at roomtemperature. Plates were washed with PBS after each step.

For competition assays, binding of [¹²⁵I]-HGF (specific activity ˜2,000Ci/mmol) at 200 μM to immobilized c-Met was measured in the presence ofvarying concentrations of the anti-c-Met monoclonal antibodies 11E1,224G11, 223C4, 227H1 or HGF (R&D Systems) ranging from 0.1 pM to 1 μM inPBS pH 7.4. The plates were incubated at room temperature for 6 h, thencounted on a Packard Top Count Microplate Scintillation Counter. Nonspecific binding was determined in the presence of 1 μM of HGF. Themonoclonal antibody 9G4, which is not directed at c-Met but specificallyrecognizes an E. coli protein, was used as mouse IgG1 isotype control.

Percent of total specific [¹²⁵I]-HGF binding was plotted as a functionof ligand concentration on semilog graphs. Concentrations of the variousinhibitors required to inhibit the radioligand binding by 50% (IC₅₀)were determined graphically from the sigmoid competition curves obtained(FIGS. 7A and 7B).

As expected, non radiolabeled HGF was able to fully displace [¹²⁵I]-HGFbinding to immobilized c-Met, whereas the control antibody 9G4 did notshow any HGF blocking activity (FIGS. 7A and 7B). Monoclonal anti-c-Metantibodies 11E1, 224G11, 223C4 and 227H1 were able to inhibit [¹²⁵I]-HGFbinding to immobilized c-Met, with IC₅₀ values of 20 nM, 3 nM, 2.7 nMand 5.8 nM, respectively. The IC₅₀ values determined for antibodies224G11, 223C4 and 227H1 were comparable to the IC₅₀ value determined fornon radiolabeled HGF, which was comprised between 3 and 5 nM, whereasantibody 11E1 exhibited a higher IC₅₀ value.

EXAMPLE 5 Inhibition of Invasion by Anti-c-Met Antibodies

To evaluate the inhibiting effect of the anti-c-Met antibodies on theinvasion process, A549 cells were plated in the upper chamber of BDBioCoat™ Matrigel™ invasion chambers (6.5 mm diameter wells with 8-μmpre size polycarbonate membranes). A459 cells were starved 24 hoursbefore performing the invasion assay. Then 500 000 A549 cells wereplated in chemotaxis buffer (DMEM medium, 0.1% BSA, 12 mM Hepes) in theupper well of each chamber, upon the Matrigel coating either with orwithout the antibody to be tested (final Mab concentration10 μg/ml).After 1 hour incubation of the plates at 37° C. with 5% CO₂, the lowerchambers were filled with either growth medium containing 400 ng/ml ofrhHGF or with growth medium alone. The chambers were incubated for 48additional hours at 37° C. with 5% CO2. At the end of this incubationtime, cells that remained on upper surface of the filter were gentlyremoved with a cotton swab, cells that migrated to the lower surface ofthe filter were lysed, stained with CyQuant GR dye buffer (Invitrogen)and counted using a fluorescence reader Berthold Mithras LB940. Allconditions were tested as triplicates.

As expected HGF induced a significant invasion of tumor cells comparableto the one observed with 10% FCS introduced as a positive control (FIG.8). The murine IgG1 9G4 introduced as an isotype control is withoutsignificant effect on basal or HGF-induced invasion when compared tocells plated without IgG. No agonist effect was noticed with 11E1,224G11, 223C4 and 227H1 when added alone and a significant andcomparable inhibition of the HGF-induced invasion was observed with the3 Mabs.

EXAMPLE 6 Inhibition of Wound Healing by Anti-c-Met Antibodies

HGF stimulates motility. To determine whether the anti-HGF antibodieswere able to inhibit migration, NCI-H441 cells were grown to highdensity and a gap was introduced with a P200 pipette tip. Cells werethen stimulate to migrate across the gap with HGF (100 ng/ml) inpresence or in absence of 11E1. Wells with 11E1 alone were alsoevaluated. Each tested condition was evaluated as a sextuplicate and 3independent experiments were performed. After an overnight incubation,cells were visualized with an Axio Vision Camera (objective ×4).

HGF induced a significant migration resulting in a complete closure ofthe gad within one night (FIG. 9). The 9G4 irrelevant IgG1 used as anisotype control is without any effect on cell migration. As expected anagonist effect was observed with the 5D5 when added alone but asignificant inhibition of cell migration is observed with this antibodyin presence of HGF in the portion of the gap remained open. The Fabfragment of 5D5 is without any agonist effect when added alone. Howeverno activity of this fragment was observed in presence of HGF. Asobserved with the isotype control 9G4, the MAb 11E1 had no agonisteffect when added alone and behave as a full antagonist in presence ofHGF.

EXAMPLE 7 Scatter Assay

SK-HEP-1 cells were seeded at low density (1.10⁴ cells/well) in a24-well plate in DMEM with 10% FCS and grown for 24 hours beforeaddition, at the same time, of HGF (100 ng/ml) and antibodies to betested (10 ng/ml). After 72 hours incubation, colonies were fixed andstained with 0.2% crystal violet in methanol and assessed for scatteringvisually. Each tested condition was tested as a triplicate and 3independent experiments were performed.

Addition of HGF to SK-HEP-1 cells induced a significant cell scattering(FIGS. 10A and 10B). The 9G4 antibody introduced as an isotype controlis without effect neither alone or in presence of HGF. As expected the5D5 antibody displayed a significant agonist effect alone and noinhibitory effect was observed when 5D5 was added with HGF (FIG. 10A).No agonistic effect was observed neither with 11E1 (FIG. 10A) nor with224G11 (FIG. 10B) added alone. A very significant inhibitory effect ofthese antibodies was demonstrated in presence of HGF (FIGS. 10A and10B).

EXAMPLE 8 Three-Dimensional Tubulogenesis Assay

SK-HEP-1 cells were seeded at 1.10⁴ cells/well in a 24-well plate inDMEM with 10% FCS/Matrigel (50/50) and incubated for 30 min beforeaddition, at the same time, of HGF (100 ng/ml) and antibodies to betested (10 ng/ml). After 7 days incubation, cells were assessed for tubeformation visually. Each tested condition was tested as a triplicate and3 independent experiments were performed.

Addition of HGF induced a significant SK-HEP-1 tube formation (FIG. 11).The antibody 9G4 introduced as an isotype control was without effectneither alone or in presence of HGF. As expected the 5D5 antibodydisplayed a significant agonist effect alone and no inhibitory effectwas observed when 5D5 was added with HGF. No agonistic effect wasobserved with 11E1, 223C4 and 224G11 added alone and a full inhibitoryeffect was demonstrate with both 11E1 and 223C4 in presence of HGF. Apartial but significant inhibition was observed with the 224G11Mab.

EXAMPLE 9 Spheroid Formation

To evaluate the ability of anti-c-Met antibodies to inhibit in vitrotumor growth, in a model closer to an in vivo situation, U-87MG, humanglioblastoma cells (ATCC #HTB-14) spheroids were generated. Cells grownas a monolayer were detached with trypsine-EDTA and resuspended intocomplete cell culture media (DMEM) supplemented with 10% FBS. Spheroidswere initiated by inoculating 625 cells into single wells of roundbottom, 96 plates in DMEM-10% FCS. To prohibit cell adhesion to asubstratum, the plates were pre-coated with polyHEMA in 95% ethanol andair dried at room temperature. The plates were incubated under standardcell culture conditions at 37° C., 5% CO2 in humidified incubators.Purified monoclonal antibodies (10 μg/ml) were added after 3 and 7 daysof spheroid culture. HGF (400 ng/ml) was added once after 4 days ofculture. Spheroids were kept in culture for at least 10 days. Then,spheroid growth was monitored by measuring the area of spheroids usingautomeasure module of axiovision software. Area was expressed in μm².8-16 spheroids were evaluated for each condition.

FIGS. 12A and 12B showed that in presence of 10% FCS no stimulation wasobserved when HGF was added to the complete medium. As expected the 9G4isotype control is without effect on spheroid growth. 11E1 and 223C4reduced significantly spheroid growth both in presence and in absence ofHGF. No effect was observed with the 5D5 Fab fragment.

EXAMPLE 10 In Vivo Activity of Anti-c-Met Mabs in the U87MG XenograftModel

Six to eight weeks old athymic mice were housed in sterilizedfilter-topped cages, maintained in sterile conditions and manipulatedaccording to French and European guidelines. U87-MG, a glioblastoma cellline, expressing c-Met and autocrine for the ligand HGF, was selectedfor in vivo evaluations. Mice were injected subcutaneously with 5×10⁶cells. Then, six days after cell implantation, tumors were measurable(approximately 100 mm³), animals were divided into groups of 6 mice withcomparable tumor size and treated twice a week with 1 mg/dose of eachantibody to be tested. The mice were followed for the observation ofxenograft growth rate and body weight changes. Tumor volume wascalculated by the formula: π(Pi)/6×length×width×height.

The results obtained were summarized in FIG. 13 and demonstrated thatall tested antibodies inhibit significantly in vivo growth of U87-MGcells. The use of a neutralizing anti-IGF-1R antibody (IgG1) in panel Ademonstrates that the observed in vivo inhibition is specificallyrelated to a HGF-cMet axis modulation.

EXAMPLE 11 In Vivo Activity of Anti-c-Met Mabs in the NCI-H441 XenograftModel

NCI-H441 is derived from papillary lung adenocarcinoma, expresses highlevels of c-Met, and demonstrates constitutive phosphorylation of c-MetRTK.

To determine whether this cell line expresses high levels of c-Met andis able to produce HGF, both quantitative RT-PCRs and FACS or ELISA(Quantikine HGF; R&D systems) were performed. For quantitative RT-PCRs,total HGF or cMet transcript expression levels in cell lines wereassessed by quantitative PCR using standard TaqMan™ technique. HGF orc-Met transcript levels were normalized to the housekeeping geneRibosomal protein, large, P0(RPL0) and results were expressed asnormalized expression values (2-ddCT method).

The primer/probe sets for RPL0 were forward,5′-gaaactctgcattctcgcttectg-3′ (SEQ ID No. 47); reverse,5′-aggactcgtttgtacccgttga-3′ (SEQ ID No. 48); and probe,5′-(FAM)-tgcagattggctacccaactgttgca-(TAMRA)-3′ (SEQ ID No. 49). Theprimer/probe sets for HGF were forward, 5′-aacaatgcctctggttcc-3′ (SEQ IDNo. 50); reverse, 5′-cttgtagagcgtcctttac-3′ (SEQ ID No. 51); and probe,5′-(FAM)-ccttcaatagcatgtcaagtggagtga-(TAMRA)-3′ (SEQ ID No. 52). Theprimer/probe sets for cMet were forward,5′-cattaaaggagacctcaccatagctaat-3′ (SEQ ID No. 53); reverse,5′-cctgatcgagaaaccacaacct-3′ (SEQ ID No. 54); and probe,5′-(FAM)-catgaagcgaccctctgatgtccca-(TAMRA)-3′ (SEQ ID No. 55). Thethermocycling protocol consisted of melting at 50° C. for 2 minutes and95° C. for 10 minutes, followed by 40 cycles at 95° C. for 15 secondsand 62° C. for 1 minute.

No mRNA for HGF was found in NCI-H441 (FIG. 14) and HGF is notdetectable by ELISA in NCI-H441 supernatants. In these experimentsU87-MG, a glioblastoma cell line known as an autocrine cell line forHGF, was introduced as a positive control. The RT-PCR analysis showed asignificant level of HGF mRNA in U87-MG and 1.9 ng HGF/million cells wasdetected in the supernatant of U87-MG cells. Both quantitative RT-PCRsand FACS analysis FIGS. 15A and 15B demonstrated that as expectedNCI-H441 cells significantly overexpressed c-Met and that thisexpression was dramatically higher than the one observed for U87-MGcells. In this experiment the MCF-7 cell line was introduced as anegative control. Taken together NCI-H441 appears as a non autocrineconstitutively activated cell line able to grow independently of HGFligand in which a ligand-independent dimerization of c-met occurred as aconsequence of the overexpression of the receptor.

The evaluation of anti-c-met antibodies on the in vivo activity of thisnon autocrine cell line could give some insights about their potency toimpact on c-met dimerization.

FIG. 16 demonstrates that 224G11, 11E1 and 227H1 inhibited significantlyin vivo growth of NCI-H441 suggesting that in addition to liganddependent inhibition, these antibodies able to inhibit dimerization arealso able to target a ligand-independent inhibition of c-met. Asmentioned above in the specification, with that last property, 224G11,11E1 and 227H1 are shown to be different from the 5D5 one armed (OA-5D5)anti-c-Met antibody.

EXAMPLE 12 Humanization Process by CDR-Grafting of the Antibody 224G11I—Humanization of the Light Chain Variable Domain

Comparison of the Nucleotidic Sequence of the 224G11VL with MurineGermline Genes

As a preliminary step, the nucleotidic sequence of the 224G11 VL wascompared to the murine germline genes sequences part of the IMGTdatabase (http://imgt.cines.fr).

Murine IGKV3-5*01 and IGKJ4*01 germline genes with a sequence identityof 99.31% for the V region and 94.28% for the J region, respectively,have been identified. Regarding the obtained identity, it has beendecided to directly use the 224G11VL sequences to look for humanhomologies.

These alignments are represented in FIGS. 17A for the V gene and 17B forthe J gene.

Comparison of the Nucleotidic Sequence of the 224G11VL with HumanGermline Genes

In order to identify the best human candidate for the CDR grafting, thehuman germline gene displaying the best identity with the 224G11VL hasbeen searched. To this end, the nucleotidic sequence of 224G11VL hasbeen aligned with the human germline genes sequences part of the IMGTdatabase. For optimization of the selection, alignments between theproteic sequences were made to search for better homologies.

These two complementary methods led to the identification of twopossible receiving human V sequences for the murine 224G11 VL CDRs.Nucleotidic alignment gives the human IGKV3-11*01 germline gene with asequence identity of 75.99% whereas proteic alignment gives the humanIGKV4-1*01 germline gene with a sequence identity of 67.30%. Itworthnoting that in both cases, the two closest germline genes and theanalysed sequences show different CDR1 amino acid lengths (10 aminoacids in 224G11 VL; 6 amino acids in IGKV3-11*01; 12 amino acids inIGKV4-1*01).

For the J region, the best homology score was first obtained with humanthe human IGKJ3*01 showing a sequence identity of 80%. But a highernumber of consecutive identical nucleotides and a better amino acidfitting has been found in the alignment with human IGKJ4*02 germlinegene (sequence identity of 77.14%). Thus the IGKJ4*02 germline gene wasselected as receiving human J region for the murine 11E1 VL CDRs.

Alignments are represented in FIGS. 18A for the V region and 18B for theJ region.

Humanized Version of 224G11 VL

Given the possibility of two receiving human V regions for the murine224G11 VL CDRs, two humanized versions of the 224G11 VL domain will bedescribed. The first corresponds to an initial trial for a humanframework with a shorter CDR1 length (IGKV3-11*01), the second with alonger CDR1 length (IGKV4-1*01).

a) IGKV3-11*01 Based Humanized Version of 224G11 VL

The following steps in the humanization process consist in linking theselected germline genes sequences IGKV3-11*01 and IGKJ4*02 and also theCDRs of the murine 224G11 VL to the frameworks of these germline genessequences.

As depicted in FIG. 19A, the bolded residues in the 224G11 VL sequencecorrespond to the twenty-five amino acids that were found differentbetween 224G11 VL domain and the selected human frameworks (Human FR,i.e. IGKV3-11*01 and IGKJ4*02).

Regarding to several criteriae such as their known participation inVH/VL interface, in antigen binding or in CDR structure, the amino acidclass changes between murine and human residues, localization of theresidue in the 3D structure of the variable domain, three out of thetwenty-five different residues have been identified to be eventuallymutated. These three most important defined residues and mutations intotheir human counterparts being murine M39 into human L, H40 into A andR84 into G. These ranked one residues are shown in FIG. 19A as boldedresidues in the 224G11 HZ1VL sequence where they remained murine.

Of course, the above mentioned residues to be tested are not limited butmust be considered as preferential mutations.

With the help of a molecular model, other mutations could be identified.Can be mentioned the following ranked two residues, i.e. residues 15(L/P), 49 (P/A), 67 (L/R), 68 (E/A), 93 (P/S) and 99 (V/F) on whichmutations could also be envisaged in another preferred embodiment.

Of course, the above mentioned residues to be eventually tested are notlimited but must be considered as preferential mutations. In anotherpreferred embodiment, all the sixteen others ranked three residues amongthe twenty-five different amino acids could be reconsidered.

All the above mentioned mutations will be tested individually oraccording various combinations.

FIG. 19A represents the implemented IGKV3-11*01 based humanized 224G11VL with above mentioned mutations clearly identified. The number undereach proposed mutation corresponds to the rank at which said mutationwill be done.

b) IGKV4-1*01 Based Humanized Version of 224G11 VL

The following steps in the humanization process consist in linking theselected germline genes sequences IGKV4-1*01 and IGKJ4*02 and also theCDRs of the murine 224G11 VL to the frameworks of these germline genessequences.

As depicted in FIG. 19B, the bolded residues in the 224G11 VL sequencecorresponds to the twenty-two amino acids that were found differentbetween 224G11 VL domain and the selected human frameworks (Human FR,i.e. IGKV4-1*01 and IGKJ4*02).

Regarding to several criteriae such as their known participation inVH/VL interface, in antigen binding or in CDR structure, the amino acidclass changes between murine and human residues, localization of theresidue in the 3D structure of the variable domain, four out of thetwenty-two different residues have been identified to be eventuallymutated. These four most important defined residues and mutations intotheir human counterparts being murine L4 into human M, M39 into L, H40into A and R84 into G. These ranked one residues are shown in FIG. 19Bas bolded residues in the 224G11 HZ2VL sequence where they remainedmurine.

Of course, the above mentioned residues to be tested are not limited butmust be considered as preferential mutations.

With the help of a molecular model, other mutations could be identified.Can be mentioned the following ranked two residues, i.e. residues 25(A/S), 66 (N/T), 67 (L/R), and 93 (P/S) on which mutations could also beenvisaged in another preferred embodiment.

Of course, the above mentioned residues to be eventually tested are notlimited but must be considered as preferential mutations. In anotherpreferred embodiment, all the fourteen others ranked three residuesamong the twenty-two different amino acids could be reconsidered.

All the above mentioned mutations will be tested individually oraccording various combinations.

FIG. 19B represents the implemented IGKV4-1*01 based humanized 224G11 VLwith above mentioned mutations clearly identified. The number under eachproposed mutation corresponds to the rank at which said mutation will bedone.

II—Humanization of the Heavy Chain Variable Domain

Comparison of the nucleotidic sequence of the 224G11 VH with murinegermline genes

As a preliminary step, the nucleotidic sequence of the 224G11 VH wascompared to the murine germline genes sequences part of the IMGTdatabase (http://imgt.cines.fr).

Murine IGHV1-18*01, IGHD2-4*01 and IGHJ2*01 germline genes with asequence identity of 92.70% for the V region, 75.00% for the D regionand 89.36% for the J region, respectively, have been identified.Regarding the obtained identity, it has been decided to directly use the224G11 VH sequences to look for human homologies.

These alignments are represented in FIGS. 20A for the V gene, 20B forthe D gene and 20C for the J gene.

Comparison of the Nucleotidic Sequence of the 224G11 VH with HumanGermline Genes

In order to identify the best human candidate for the CDR grafting, thehuman germline gene displaying the best identity with the 224G11 VH hasbeen searched. To this end, the nucleotidic sequence of 224G11 VH hasbeen aligned with the human germline genes sequences part of the IMGTdatabase. For optimization of the selection, alignments between theproteic sequences were made to search for better homologies.

These two complementary methods led to the identification of the samereceiving human IGHV1-2*02 V sequence for the murine 224G11 VH CDRs witha sequence identity of 75.00% at the nucleotidic level and 64.30% at theproteic level.

It is worthnoting that the D region strictly belongs to the CDR3 regionin the VH domain. The humanization process is based on a<<CDR-grafting>> approach. Analysis of the closest human D-genes is notuseful in this strategy.

Looking for homologies for the J region led to the identification of thehuman IGHJ4*04 germline gene with a sequence identity of 78.72%.

Human IGHV1-2*02 V germline gene and human IGHJ4*01 J germline gene havethus been selected as receiving human sequences for the murine 224G11 VHCDRs.

Alignments are represented in FIG. 21A for the V regionand 21B for the Jregion.

Humanized Version of 224G11 VH

The following steps in the humanization process consist in linking theselected germline genes sequences IGHV1-2*02 and IGHJ4*01 and also theCDRs of the murine 224G11 VH to the frameworks of these germline genessequences.

As depicted in FIG. 22, the bolded residues in the 224G11 VH sequencecorrespond to the thirty amino acids that were found different between224G11 VH domain and the selected human frameworks (Human FR, i.e.IGHV1-2*02 and IGHJ4*01).

Regarding to several criteriae such as their known participation inVH/VL interface, in antigen binding or in CDR structure, the amino acidclass changes between murine and human residues, localization of theresidue in the 3D structure of the variable domain, four out of thethirty different residues have been identified to be eventually mutated.These four most important defined residues and mutations into theirhuman counterparts being murine D51 into human E, G55 into W, V80 into Rand K82 into T. These ranked one residues are shown in FIG. 22 as boldedresidues in the 224G11 HZVH sequence where they remained murine.

Of course, the above mentioned residues to be tested are not limited butmust be considered as preferential mutations.

With the help of a molecular model, other mutations could be identified.Can be mentioned the following ranked two residues, i.e. residues 25(T/A), 48 (E/Q), 49 (S/G), 53 (I/M), 76 (A/V), 78 (L/M) and 90 (D/E) onwhich mutations could also be envisaged in another preferred embodiment.

Of course, the above mentioned residues to be eventually tested are notlimited but must be considered as preferential mutations. In anotherpreferred embodiment, all the nineteen others ranked three residuesamong the thirty different amino acids could be reconsidered.

All the above mentioned mutations will be tested individually oraccording various combinations.

FIG. 22 represents the humanized 224G11 VH with above mentionedmutations clearly identified. The number under each proposed mutationcorresponds to the rank at which said mutation will be done.

EXAMPLE 13 Humanization Process by CDR-Grafting of the Antibody 227H1

I—Humanization of the Light Chain Variable Domain

Comparison of the nucleotidic sequence of the 227H1 VL with murinegermline genes

As a preliminary step, the nucleotidic sequence of the 227H1 VL wascompared to the murine germline genes sequences part of the IMGTdatabase (http://imgt.cines.fr).

Murine IGKV3-5*01 and IGKJ4*01 germline genes with a sequence identityof 96.90% for the V region and 97.29% for the J region, respectively,have been identified. Regarding the obtained identity, it has beendecided to directly use the 227H1 VL sequences to look for humanhomologies.

These alignments are represented in FIGS. 23A for the V gene and 23B forthe J gene.

Comparison of the Nucleotidic Sequence of the 227H1 VL with HumanGermline Genes

In order to identify the best human candidate for the CDR grafting, thehuman germline gene displaying the best identity with the 227H1 VL hasbeen searched. To this end, the nucleotidic sequence of 227H1 VL hasbeen aligned with the human germline genes sequences part of the IMGTdatabase. For optimization of the selection, alignments between theproteic sequences were made to search for better homologies.

These two complementary methods led to the identification of twopossible receiving human V sequences for the murine 227H1 VL CDRs.Nucleotidic alignment gives the human IGKV3-11*01 germline gene with asequence identity of 7491% whereas proteic alignment gives the humanIGKV4-1*01 germline gene with a sequence identity of 64.00%. Itworthnoting that in both cases, the two closest germline genes and theanalysed sequences show different CDR1 amino acid lengths (10 aminoacids in 227H1 VL; 6 amino acids in IGKV3-11*01; 12 amino acids inIGKV4-1*01).

For the J region, the best homology score was first obtained with humanthe human IGKJ3*01 showing a sequence identity of 78.38%. But a highernumber of consecutive identical nucleotides and a better amino acidfitting has been found in the alignment with human IGKJ4*02 germlinegene (sequence identity of 75.68%). Thus the IGKJ4*02 germline gene wasselected as receiving human J region for the murine 227H1 VL CDRs.

Alignments are represented in FIGS. 24A for the V region and 24B for theJ region.

Humanized Version of 224G11 VL

Given the possibility of two receiving human V regions for the murine227H1 VL CDRs, two humanized versions of the 227H1 VL domain will bedescribed. The first corresponds to an initial trial for a humanframework with a shorter CDR1 length (IGKV3-11*01), the second with alonger CDR1 length (IGKV4-1*01).

a) IGKV3-11*01 Based Humanized Version of 227H1 VL

The following steps in the humanization process consist in linking theselected germline genes sequences IGKV3-11*01 and IGKJ4*02 and also theCDRs of the murine 227H1 VL to the frameworks of these germline genessequences.

As depicted in FIG. 25A, the bolded residues in the 227H1 VL sequencecorresponds to the twenty-six amino acids that were found differentbetween 227H1 VL domain and the selected human frameworks (Human FR,i.e. IGKV3-11*01 and IGKJ4*02).

Regarding to several criteriae such as their known participation inVH/VL interface, in antigen binding or in CDR structure, the amino acidclass changes between murine and human residues, localization of theresidue in the 3D structure of the variable domain, three out of thetwenty-six different residues have been identified to be eventuallymutated. These three most important defined residues and mutations intotheir human counterparts being murine 139 into human L, H40 into A andR84 into G. These ranked one residues are shown in FIG. 25A as boldedresidues in the 227H1 HZ1VL sequence where they remained murine.

Of course, the above mentioned residues to be tested are not limited butmust be considered as preferential mutations.

With the help of a molecular model, other mutations could be identified.Can be mentioned the following ranked two residues, i.e. residues 15(L/P), 25 (V/A), 49 (P/A), 67 (L/R), 68 (E/A), 93 (P/S) and 99 (S/F) onwhich mutations could also be envisaged in another preferred embodiment.

Of course, the above mentioned residues to be eventually tested are notlimited but must be considered as preferential mutations. In anotherpreferred embodiment, all the sixteen others ranked three residues amongthe twenty-five different amino acids could be reconsidered.

All the above mentioned mutations will be tested individually oraccording various combinations.

FIG. 25A represents the implemented IGKV3-11*01 based humanized 227H1 VLwith above mentioned mutations clearly identified. The number under eachproposed mutation corresponds to the rank at which said mutation will bedone.

b) IGKV4-1*01 Based Humanized Version of 227H1 VL

The following steps in the humanization process consist in linking theselected germline genes sequences IGKV4-1*01 and IGKJ4*02 and also theCDRs of the murine 227H1 VL to the frameworks of these germline genessequences.

As depicted in FIG. 25B, the bolded residues in the 227H1 VL sequencecorresponds to the twenty-four amino acids that were found differentbetween 227H1 VL domain and the selected human frameworks (Human FR,i.e. IGKV4-1*01 and IGKJ4*02).

Regarding to several criteriae such as their known participation inVH/VL interface, in antigen binding or in CDR structure, the amino acidclass changes between murine and human residues, localization of theresidue in the 3D structure of the variable domain, four out of thetwenty-four different residues have been identified to be eventuallymutated. These four most important defined residues and mutations intotheir human counterparts being murine L4 into human M, 139 into L, H40into A and R84 into G. These ranked one residues are shown in FIG. 25Bas bolded residues in the 227H1 HZ2VL sequence where they remainedmurine.

Of course, the above mentioned residues to be tested are not limited butmust be considered as preferential mutations.

With the help of a molecular model, other mutations could be identified.Can be mentioned the following ranked two residues, i.e. residues 25(V/S), 66 (N/T), 67 (L/R), and 93 (P/S) on which mutations could also beenvisaged in another preferred embodiment.

Of course, the above mentioned residues to be eventually tested are notlimited but must be considered as preferential mutations. In anotherpreferred embodiment, all the sixteen others ranked three residues amongthe twenty-two different amino acids could be reconsidered.

All the above mentioned mutations will be tested individually oraccording various combinations.

FIG. 25B represents the implemented IGKV4-1*01 based humanized 227H1 VLwith above mentioned mutations clearly identified. The number under eachproposed mutation corresponds to the rank at which said mutation will bedone.

II—Humanization of the Heavy Chain Variable Domain

Comparison of the nucleotidic sequence of the 227H1 VH with murinegermline genes

As a preliminary step, the nucleotidic sequence of the 227H1 VH wascompared to the murine germline genes sequences part of the IMGTdatabase (http://imgt.cines.fr).

Murine IGHV1-18*01, IGHD1-1*02 and IGHJ2*01 germline genes with asequence identity of 92.70% for the V region, 63.63% for the D regionand 91.48% for the J region, respectively, have been identified.Regarding the obtained identity, it has been decided to directly use the227H1 VH sequences to look for human homologies.

These alignments are represented in FIGS. 26A for the V gene, 26B forthe D gene and 26C for the J gene.

Comparison of the Nucleotidic Sequence of the 227H1 VH with HumanGermline Genes

In order to identify the best human candidate for the CDR grafting, thehuman germline gene displaying the best identity with the 224G11 VH hasbeen searched. To this end, the nucleotidic sequence of 227H1 VH hasbeen aligned with the human germline genes sequences part of the IMGTdatabase. The receiving human IGHV1-2*02 V sequence for the murine224G11 VH CDRs with a sequence identity of 72.92% was thus identified.

It is worthnoting that the D region strictly belongs to the CDR3 regionin the VH domain. The humanization process is based on a<<CDR-grafting>> approach. Analysis of the closest human D-genes is notuseful in this strategy.

Looking for homologies for the J region led to the identification of thehuman IGHJ4*01 germline gene with a sequence identity of 78.72%.

Human IGHV1-2*02 V germline gene and human IGHJ4*01 J germline gene havethus been selected as receiving human sequences for the murine 227H1 VHCDRs.

Alignments are represented in FIGS. 27A for the V region and 27B for theJ region.

For optimisation of the selection, the man skilled in the art could alsomake alignments between the proteic sequences in order to help him inthe choice.

Humanized Version of 227H1 VH

The following steps in the humanization process consist in linking theselected germline genes sequences IGHV1-2*02 and IGHJ4*01 and also theCDRs of the murine 227H1 VH to the frameworks of these germline genessequences.

As depicted in FIG. 28, the bolded residues in the 227H1 VH sequencecorrespond to the thirty-two amino acids that were found differentbetween 227H1 VH domain and the selected human frameworks (Human FR,i.e. IGHV1-2*02 and IGHJ4*01).

Regarding to several criteriae such as their known participation inVH/VL interface, in antigen binding or in CDR structure, the amino acidclass changes between murine and human residues, localization of theresidue in the 3D structure of the variable domain, six out of thethirty-two different residues have been identified to be eventuallymutated. These six most important defined residues and mutations intotheir human counterparts being murine L39 into human M, N40 into H, L55into W, T66 into N, V80 into R and K82 into T. These ranked one residuesare shown in FIG. 28 as bolded residues in the 227H1 HZVH sequence wherethey remained murine.

Of course, the above mentioned residues to be tested are not limited butmust be considered as preferential mutations.

With the help of a molecular model, other mutations could be identified.Can be mentioned the following ranked two residues, i.e. residues 48(K/Q), 49 (T/G), 53 (UM), 76 (A/V) and 78 (L/M) on which mutations couldalso be envisaged in another preferred embodiment.

Of course, the above mentioned residues to be eventually tested are notlimited but must be considered as preferential mutations. In anotherpreferred embodiment, all the twenty-one others ranked three residuesamong the thirty different amino acids could be reconsidered.

All the above mentioned mutations will be tested individually oraccording various combinations.

FIG. 28 represents the humanized 227H1 VH with above mentioned mutationsclearly identified. The number under each proposed mutation correspondsto the rank at which said mutation will be done.

EXAMPLE 14 Humanization Process by CDR-Grafting of the Antibody 223C4

I—Humanization of the Light Chain Variable Domain

Comparison of the Nucleotidic Sequence of the 223C4 VL with MurineGermline Genes

As a preliminary step, the nucleotidic sequence of the 223C4 VL wascompared to the murine germline genes sequences part of the IMGTdatabase (http://imgt.cines.fr).

Murine IGKV12-46*01 and IGKJ2*01 germline genes with a sequence identityof 99.64% for the V region and 94.59% for the J region, respectively,have been identified. Regarding the obtained identity, it has beendecided to directly use the 223C4 VL sequences to look for humanhomologies.

These alignments are represented in FIGS. 29A for the V gene and 29B forthe J gene.

Comparison of the Nucleotidic Sequence of the 223C4 VL with HumanGermline Genes

In order to identify the best human candidate for the CDR grafting, thehuman germline gene displaying the best identity with the 223C4 VL hasbeen searched. To this end, the nucleotidic sequence of 223C4 VL hasbeen aligned with the human germline genes sequences part of the IMGTdatabase.

Human IGKV1-NL1*01 and IGKJ2*01 germline genes with a sequence identityof 78.49% for the V region and 81.08% for the J region, respectively,have been identified. The germline genes IGKV1-NL1*01 for the V regionand IGKJ2*01 for the J region have thus been selected as receiving humansequences for the murine 223C4 VL CDRs.

Alignments are represented in FIGS. 30A for the V region and 30B for theJ region.

For optimisation of the selection, the man skilled in the art could alsomake alignments between the proteic sequences in order to help him inthe choice.

Humanized Version of 223C4 VL

The following steps in the humanization process consist in linking theselected germline genes sequences IGKV1-NL1*01 and IGKJ2*01 and also theCDRs of the murine 223C4 VL to the frameworks of these germline genessequences.

At this stage of the process, a molecular model of the 223C4 murine Fvdomains could be developed and useful in the choice of the murineresidues to be conserved due to their roles in the maintenance of thethree-dimensional structure of the molecule or in the antigen bindingsite and function. More particularly, 9 residues to be eventuallymutated have been identified.

In a first step, residues involved in the CDR anchors or structure willbe tested. Such residues are residue 66 (R/N) and residue 68 (E/V).

In a second step, residues exposed to solvent, and as such that mayinvolve immunogenicity, will also be tested. These are residues 49(A/S), 51 (K/Q), 69 (S/D), 86 (D/Q) and 92 (S/N).

Then, in a third step, residues involved in structure/folding ofvariable domain could also be mutated. These residues are residue 46(P/Q) and residue 96 (P/S).

Of course, the above mentioned residues to be tested are not limited butmust be considered as preferential mutations.

With the help of a molecular model, other mutations could be identified.Can be mentioned the following residues, i.e. residues 9 (S/A), 13(A/V), 17 (D/E), 18 (R/T), 54 (L/V), 88 (T/S), 90 (T/K), 100 (A/G) and101 (T/S), on which mutations could also be envisaged in anotherpreferred embodiment.

All the above mentioned mutations will be tested individually oraccording various combinations.

FIG. 31 represents the humanized 223C4 VL with above mentioned mutationsclearly identified. The number under each proposed mutation correspondsto the rank at which said mutation will be done.

II—Humanization of the Heavy Chain Variable Domain

Comparison of the Nucleotidic Sequence of the 223C4 VH with MurineGermline Genes

As a preliminary step, the nucleotidic sequence of the 223C4 VH wascompared to the murine germline genes sequences part of the IMGTdatabase (http://imgt.cines.fr).

Murine IGHV1-18*01, IGHD6-3*01 and IGHJ4*01 germline genes with asequence identity of 98.95% for the V region, 72.72% for the D regionand 98.11% for the J region, respectively, have been identified.Regarding the obtained identity, it has been decided to directly use the223C4 VH sequences to look for human homologies.

These alignments are represented in FIGS. 32A for the V gene, 32B forthe D gene and 32C for the J gene.

Comparison of the Nucleotidic Sequence of the 223C4 VH with HumanGermline Genes

In order to identify the best human candidate for the CDR grafting, thehuman germline gene displaying the best identity with the 223C4 VH hasbeen searched. To this end, the nucleotidic sequence of 223C4 VH hasbeen aligned with the human germline genes sequences part of the IMGTdatabase.

Human IGHV1-2*02, IGHD1-26*01 and IGHJ6*01 germline genes with asequence identity of 76.38% for the V region, 75.00% for the D regionand 77.41% for the J region, respectively, have been identified. Thegermline genes IGHV1-2*02 for the V region and IGHJ6*01 for the J regionhave thus been selected as receiving human sequences for the murine223C4 VH CDRs.

Alignments are represented in FIGS. 33A for the V region, 33B for the Dregion and 33C for the J region.

For optimisation of the selection, the man skilled in the art could alsomake alignments between the proteic sequences in order to help him inthe choice.

Humanized Version of 223C4 VH

The following steps in the humanization process consist in linking theselected germline genes sequences IGHV1-2*02 and IGHJ6*01 and also theCDRs of the murine 223C4 VH to the frameworks of these germline genessequences.

At this stage of the process, a molecular model of the 223C4 murine Fvdomains could be developed and useful in the choice of the murineresidues to be conserved due to their roles in the maintenance of thethree-dimensional structure of the molecule or in the antigen bindingsite and function. More particularly, 14 residues to be eventuallymutated have been identified.

In a first step, residues involved in the CDR anchors or structure willbe tested. Such residues are residues 40 (H/D), 45 (A/S), 55 (W/D), 66(N/I) and 67 (Y/F).

In a second step, residues exposed to solvent, and as such that mayinvolve immunogenicity, will also be tested. These are residues 1 (Q/E),3 (Q/L), 5 (V/Q), 48 (Q/M) and 80 (R/V).

Then, in a third step, residues involved in structure/folding ofvariable domain could also be mutated. These are residues 9 (A/P), 13(K/V), 22 (S/P) and 46 (P/H).

Of course, the above mentioned residues to be tested are not limited butmust be considered as preferential mutations.

With the help of a molecular model, other mutations could be identified.Can be mentioned the following residues, i.e. residues 12 (V/L), 21(V/I), 43 (R/K), 49 (G/S), 53 (M/I), 68 (A/N), 72 (Q/K), 75 (R/K), 76(V/A), 78 (M/L), 82 (T/K), 84 (I/S), 92 (S/R), 93 (R/S), 95 (R/T) and 97(D/E), on which mutations could also be envisaged in another preferredembodiment.

All the above mentioned mutations will be tested individually oraccording various combinations.

FIG. 34 represents the humanized 223C4 VH with above mentioned mutationsclearly identified. The number under each proposed mutation correspondsto the rank at which said mutation will be done.

EXAMPLE 15 Anti-Tumor Activity of the Murine 224G11 MAb Alone orCombined with the Chemotherapeutic Agent Navelbine® on the EstablishedXenograft NCI-H441 Tumor Model

Successful chemotherapeutic approaches depend in part on the cellularresponse to apoptotic inducers and the balance between pro- andanti-apoptotic pathways within the cell. The protective effect of theactivated c-Met on cell survival has been documented. It mainly resultsfrom an increase expression of the anti-apoptotic Bcl-xl and Bcl-2protein as a consequence of PI3-K-mediated signaling which in turninhibit mitochondrial-dependent apoptosis (caspase 9). Indeed, it isconceivable that the HGF/c-Met system with its marked regulatory effecton apoptotic process can also influence the chemosensitivity of cancercells. This hypothesis as been tested with Navelbine®, a marketedchemotherapeutic agent used for lung cancer treatment (Aapro et al.,Crit. Rev. Oncol. Hematol. 2001, 40:251-263; Curran et al., Drugs Aging.2002, 19:695-697). The xenograft NCI-H441 NSCLC model was used as it hasbeen previously described that this cell line is sensitive to bothNavelbine (Kraus-Berthier et al., Clin. Cancer Res., 2000; 6:297-304)and therapy targeting c-Met (Zou H. T. et al., Cancer Res. 2007, 67:4408-4417).

Briefly, NCI-H441 cells from ATCC were routinely cultured in RPMI 1640medium, 10% FCS and 1% L-Glutamine. Cells were split two days beforeengraftment so that they were in exponential phase of growth. Tenmillion NCI-H441 cells were engrafted in PBS to 7 weeks old Swiss nudemice. Three days after implantation, tumors were measured and animalswere divided into 4 groups of 6 mice with comparable tumor size. Micewere treated i.p. with a loading dose of 2 mg of 224G11/mouse and thentwice a week, for 43 days, with 1 mg of antibody/mouse. The 9G4 MAb wasused as an isotype control.

Navelbine® was given by i.p. injections at a dose of 8 mg/kg on days 5,12, 19 post-cell injection. For combined therapy with both 224G11 andNavelbine®, the two compounds were administered separately. In thisexperience the 2 compounds were used at their optimal dosage. Tumorvolume was measured twice a week and calculated by the formula:p/6×length×width×height.

FIG. 35 demonstrates that 224G11 is as efficient as Navelbine® when usedalone as a single agent therapy. A significant benefit of combining boththerapy was observed with complete tumor regressions observed for 3 outof 6 mice at day 63.

EXAMPLE 16 C-Met Inhibitors and Angiogenesis

In addition to its direct role in the regulation of a variety of tumorcell functions, activation of c-met has also been implicated in tumorangiogenesis. Endothelial cells express c-Met and HGF stimulatesendothelial cell growth, invasion and motility (Nakamura Y. et al.,Biochem. Biophys. Res., Commun 1995, 215:483-488; Bussolino F. et al.,J. Cell Biol. 1992, 119:629-641). The coordinate regulation of growth,invasion and motility in vascular endothelial cells by HGF/c-Met hasbeen demonstrated to results in the formation of 3D capillaryendothelial tubes in vitro (Rosen E. M. et al., Supplementum toExperientia 1991, 59:76-88).

To determine a potential interference of anti-c-Met MAbs withHGF-induced angiogenesis, two sets of experiments were performedincluding i) the evaluation of MAbs on HUVEC proliferation and ii) thetest of MAbs of HUVEC tube formation.

For proliferation experiments, 7500 HUVEC were plated in each well of a96 well plate previously coated with laminin. Cells were grown 24 hoursof EMB-2 assay medium supplemented with 0.5% FBS and heparin. Then, MAbsto be tested (0.15 to 40 μg/ml) were added for 1 h before addition of 20ng/ml of HGF. After 24 additional hours, cells were pulsed with 0.5 μCiof [³H] Thymidine. The magnitude of [³H] Thymidine incorporated wasquantified by liquid scintillation counting. In This experiment the 9G4MAb is an irrelevant antibody used as an IgG1 isotype control.

Results expressed as raw data in FIG. 36 demonstrate that, as expectedHGF is a potent inducer of HUVEC cell growth. Antibodies evaluated inabsence of HGF did not display any agonist proliferative activity onHUVEC whatever the tested dose. In presence of HGF, a dramatic dosedependent inhibition was observed for both 11E1 and 224G11 MAbs.

For evaluation of HUVEC tube formation, 25000 cells incubated 30 minwith antibodies to be tested were plated in 48-well plates coated withmatrigel. Then HGF 50 ng/ml was added and plates were incubated at 37°C. Medium was then harvested and 5 μM CMFDA was added for 15 min beforemicroscopic observation.

Results shown in FIG. 37 demonstrate that, as expected HGF induces asignificant tube formation. The 9G4 antibody introduced as an IgG1isotype control was without any effect on HGF-induced tube formationwhereas both 11E1 and 224G11 inhibit dramatically tube formation.

EXAMPLE 17 Humanization Process by CDR-Grafting of the Antibody 11E1

I—Humanization of the Light Chain Variable Domain

Comparison of the Nucleotidic Sequence of the 11E1 VL with MurineGermline Genes

As a preliminary step, the nucleotidic sequence of the 11E1 VL wascompared to the murine germline genes sequences part of the IMGTdatabase (http://imgt.cines.fr).

Murine IGKV4-79*01 and IGKJ4*01 germline genes with a sequence identityof 98.58% for the V region and 97.22% for the J region, respectively,have been identified. Regarding the obtained identity, it has beendecided to directly use the 11E1 VL sequences to look for humanhomologies.

These alignments are represented in FIGS. 38A for the V gene and 38B forthe J gene.

Comparison of the Nucleotidic Sequence of the 11E1 VL with HumanGermline Genes

In order to identify the best human candidate for the CDR grafting, thehuman germline gene displaying the best identity with the 11E1 VL hasbeen searched. To this end, the nucleotidic sequence of 11E1 VL has beenaligned with the human germline genes sequences part of the IMGTdatabase.

Human IGKV3-7*02 and IGKV3D-7*01 with a sequence identity for bothgermline genes of 69.86% for the V region have been identified.IGKV3-7*02 human germline gene is known in the IMGT database as an “ORF”which mean that this sequence has been found in the human genome but maypresent some recombination problems leading to non functional IGKV3-7*02derived natural antibodies. Thus the IGKV3D-7*01 germline gene wasselected as receiving human V region for the murine 11E1 VL CDRs.

For the J region, the best homology score was first obtained with human,the human IGKJ3*01 showing a sequence identity of 78.38%. But a highernumber of consecutive identical nucleotides and a better amino acidfitting has been found in the alignment with human IGKJ4*02 germlinegene (sequence identity of 75.68%). Thus the IGKJ4*02 germline gene wasselected as receiving human J region for the murine 11E1 VL CDRs.

Alignments are represented in FIGS. 39A for the V region and 39B for theJ region.

For optimisation of the selection, the man skilled in the art could alsomake alignments between the proteic sequences in order to help him inthe choice.

Humanized Version of 11E1 VL

The following steps in the humanization process consist in linking theselected germline genes sequences IGKV3D-7*01 and IGKJ4*02 and also theCDRs of the murine 11E1 VL to the frameworks of these germline genessequences.

As depicted in FIG. 40, the bolded residues in the 11E1 VL sequencecorresponds to the thirty amino acids that were found different between11E1 VL domain and the selected human frameworks (Human FR, i.e.IGKV3D-7*01 and IGKJ4*02).

Regarding to several criteriae such as their known participation inVH/VL interface, in antigen binding or in CDR structure, the amino acidclass changes between murine and human residues, localization of theresidue in the 3D structure of the variable domain, four out of thethirty different residues have been identified to be eventually mutated.These four most important defined residues and mutations into theirhuman counterparts being murine L4 into human M, Y40 into S, Y87 into Fand T96 into P. These ranked one residues are shown in FIG. 40 as boldedresidues in the 11E1 HZVL sequence where they remained murine.

Of course, the above mentioned residues to be tested are not limited butmust be considered as preferential mutations.

With the help of a molecular model, other mutations could be identified.Can be mentioned the following ranked two residues, i.e. residues 24(S/R), 53 (W/L), 66 (I/T), 67 (L/R), 86 (S/D), 95 (Q/E), 99 (A/F) or 121(E/D) on which mutations could also be envisaged in another preferredembodiment.

Of course, the above mentioned residues to be eventually tested are notlimited but must be considered as preferential mutations. In anotherpreferred embodiment, all the eighteen others ranked three residuesamong the thirty different amino acids could be reconsidered.

All the above mentioned mutations will be tested individually oraccording various combinations.

FIG. 40 represents the implemented humanized 11E1 VL with abovementioned mutations clearly identified. The number under each proposedmutation corresponds to the rank at which said mutation will be done.

II—Humanization of the Heavy Chain Variable Domain

Comparison of the Nucleotidic Sequence of the 11E1 VH with MurineGermline Genes

As a preliminary step, the nucleotidic sequence of the 11E1 VH wascompared to the murine germline genes sequences part of the IMGTdatabase (http://imgt.cines.fr).

Murine IGHV1-7*01, IGHD4-1*01 and IGHJ3*01 germline genes with asequence identity of 94.10% for the V region, 66.67% for the D regionand 100% for the J region, respectively, have been identified. Regardingthe obtained identity, it has been decided to directly use the 11E1 VHsequences to look for human homologies.

These alignments are represented in FIGS. 41A for the V gene, 41B forthe D gene and 41C for the J gene.

Comparison of the Nucleotidic Sequence of the 11E1 VH with HumanGermline Genes

In order to identify the best human candidate for the CDR grafting, thehuman germline gene displaying the best identity with the 11E1 VH hasbeen searched. To this end, the nucleotidic sequence of 11E1 VH has beenaligned with the human germline genes sequences part of the IMGTdatabase. For optimization of the selection, alignments between theproteic sequences were made to search for better homologies.

These two complementary methods led to the identification of twopossible receiving human V sequences for the murine 11E1 VH CDRs.Nucleotidic alignment gives the human IGHV1-2*02 germline gene with asequence identity of 75.69% whereas proteic alignment gives the humanIGHV1-46*01 germline gene with a sequence identity of 71.10%.

It is worthnoting that the D region strictly belongs to the CDR3 regionin the VH domain. The humanization process is based on a<<CDR-grafting>> approach. Analysis of the closest human D-genes is notuseful in this strategy.

Looking for homologies for the J region led to the identification of thehuman IGHJ4*03 germline gene with a sequence identity of 80.85%.

Looking to the overall similarities and sequences alignments, humanIGHV1-46*01 V germline gene and human IGHJ4*03 J germline gene have thusbeen selected as receiving human sequences for the murine 11E1 VH CDRs.

Alignments are represented in FIGS. 42A for the V region and 42B for theJ region.

Humanized Version of 11E1 VH

The following steps in the humanization process consist in linking theselected germline genes sequences IGHV1-46*01 and IGHJ4*03 and also theCDRs of the murine 11E1 VH to the frameworks of these germline genessequences.

As depicted in FIG. 43, the bolded residues in the 11E1 VH sequencecorresponds to the twenty-six amino acids that were found differentbetween 11E1 VH domain and the selected human frameworks (Human FR, i.e.IGHV1-46*01 and IGHJ4*03).

Regarding to several criteriae such as their known participation inVH/VL interface, in antigen binding or in CDR structure, the amino acidclass changes between murine and human residues, localization of theresidue in the 3D structure of the variable domain, five out of thetwenty-six different residues have been identified to be eventuallymutated. These five most important defined residues and mutations intotheir human counterparts being murine N40 into human H, Y55 into I, D66into S, A80 into R and K82 into T. These ranked one residues are shownin FIG. 43 as bolded residues in the 11E1 HZVH sequence where theyremained murine.

Of course, the above mentioned residues to be tested are not limited butmust be considered as preferential mutations.

With the help of a molecular model, other mutations could be identified.Can be mentioned the following ranked two residues, i.e. residues 53(I/M), 71 (L/F), 76 (A/V), 78 (L/M) and 87 (A/V) on which mutationscould also be envisaged in another preferred embodiment.

Of course, the above mentioned residues to be eventually tested are notlimited but must be considered as preferential mutations. In anotherpreferred embodiment, all the sixteen others ranked three residues amongthe twenty-six different amino acids could be reconsidered.

All the above mentioned mutations will be tested individually oraccording various combinations.

FIG. 43 represents the implemented humanized 11E1 VH with abovementioned mutations clearly identified. The number under each proposedmutation corresponds to the rank at which said mutation will be done.

EXAMPLE 18 Effect of Purified Mabs on c-Met Phosphorylation

In example 3, the effect of anti-c-Met Mabs on phosphorylation wasassessed with dosed supernatants from each hybridoma to be evaluated.The test has been performed again with purified 11E1 and 224G11 Mabsthat have been evaluated either at a final concentration of 30 μg/ml(200 nM) or at a dose range from 0.0015 to 30 μg/ml (0.01-200 nM) inorder to determine the IC₅₀ of each antibody. The protocol used is thesame as the one described in example 3.

Results of 3 independent experiments are presented in FIG. 44 anddemonstrate that once purified 11E1 and 224G11 displayed no agonisteffect when added alone to A549 cells and respectively 87 and 75%antagonist effect in presence of HGF. As expected 5D5 Mab introduced asan agonist positive control showed a significant (58%) agonist effectwhen added alone and only a moderate antagonist effect (39%) in presenceof HGF. Regarding to EC₅₀ calculations, both 11E1 and 224G11 hadnanomolar IC₅₀s.

EXAMPLE 19 In Vivo Combination of 224G11 and Navelbine® on NCI-H441Xenograft Model

NCI-H441 cells from ATCC were routinely cultured in RPMI 1640 medium,10% FCS, 1% L-Glutamine. Cells were split two days before engraftmentbeing in exponential phase of growth. Ten million NCI-H441 cells wereengrafted to Athymic nude mice. Five days after implantation, tumorswere measurable and animals were divided into groups of 6 mice withcomparable tumor size. Mice were treated i.p. either with a loading doseof 2 mg of 224G11 Mab/mouse and then twice a week with 1 mg ofantibody/mouse until Day 38 or with 3 injections of Navelbine® (D5, D12,D19) at 8 mg/kg. A third group administered with the combine treatmentwas also included. Navelbine® was given by i.p. injections. Tumor volumewas measured twice a week and calculated by the formula:π/6×length×width×height and animal weights were monitored every day overthe period of Navelbine® treatment. Statistical analysis was performedat each measured time using either a t-test or a Mann-Whitney test. Inthis experiment, the average tumor volume of single modality treatedgroups is reduced by 72%, 76% and 99.8% for 224G11, Navelbine® andNavelbine®+224G11 respectively at day 41 post first injection. At day41, the combined therapy improved significantly tumor growth compared tosingle therapy treatments (p≦041 compared to Navelbine® alone and p≦002compared to 224G11 alone on day 41), 4 out of 6 mice being withouttumors in the combined therapy group. Results are represented in FIG.45.

These results were confirmed 50 days after the end of treatments (D88)where 66% of mice receiving the combined treatment remained free oftumors.

EXAMPLE 20 In Vivo Combination of 224G11 and Doxorubicine on NCI-H441Xenograft Model

NCI-H441 cells from ATCC were routinely cultured in RPMI 1640 medium,10% FCS, 1% L-Glutamine. Cells were split two days before engraftmentbeing in exponential phase of growth. Ten million NCI-H441 cells wereengrafted to Athymic nude mice. Five days after implantation, tumorswere measurable and animals were divided into groups of 6 mice withcomparable tumor size. Mice were treated i.p. either with a loading doseof 2 mg of 224G11 Mab/mouse and then twice a week with 1 mg ofantibody/mouse or with 4 injections of Doxorubicin (D5, D12, D19, D26)at 5 mg/kg. A third group administered with the combine treatment wasalso included. Doxorubicin was given by i.v. injections. Tumor volumewas measured twice a week and calculated by the formula:m/6×length×width×height and animal weights were monitored every day overthe period of Doxorubicin treatment. Statistical analysis was performedat each measured time using either a t-test or a Mann-Whitney test. Bothsingle therapies and combined treatment displayed significant anti-tumoractivity compared to the control group (p≦002 from D11 to D39). Resultsare represented in FIG. 46.

Combined treatment also demonstrates a significant anti-tumour growthactivity compared to single modality treatment between D11 and D39indicating that there is a benefit to combine Doxorubicin to ananti-c-Met treatment.

EXAMPLE 21 In Vivo Combination of 224G11 and Docetaxel on NCI-H441Xenograft Model

NCI-H441 cells from ATCC were routinely cultured in RPMI 1640 medium,10% FCS, 1% L-Glutamine. Cells were split two days before engraftmentbeing in exponential phase of growth. Nine million NCI-H441 cells wereengrafted to Athymic nude mice. Five days after implantation, tumorswere measurable and animals were divided into groups of 6 mice withcomparable tumor size. Mice were treated i.p. either with a loading doseof 2 mg of 224G11 Mab/mouse and then twice a week with 1 mg ofantibody/mouse or with 4 injections of Docetaxel (D5, D12, D19, D26) at7.5 mg/kg. A third group administered with the combine treatment wasalso included. Docetaxel was given by i.p. injections. Tumor volume wasmeasured twice a week and calculated by the formula:π/6×length×width×height and animal weights were monitored every day overthe period of Docetaxel treatment. Statistical analysis was performed ateach measured time using either a t-test or a Mann-Whitney test. Bothsingle therapies and combined treatment displayed significant anti-tumoractivity compared to the control group (p≦0.002 from D11 to D35).Results are represented in FIG. 47.

Combined treatment also demonstrated a significant anti-tumour growthactivity compared to single modality treatment between D18 and D35indicating that there is a benefit to combine Docetaxel to an anti-c-Mettreatment.

EXAMPLE 22 In Vivo Combination of 224G11 and Temozolomide on U87MGXenograft Model

U87-MG cells from ATCC were routinely cultured in DMEM medium, 10% FCS,1% L-Glutamine. Cells were split two days before engraftment being inexponential phase of growth. Five million U87-MG cells were engrafted toAthymic nude mice. Nineteen days after implantation, tumors weremeasurable and animals were divided into groups of 6 mice withcomparable tumor size. Mice were treated i.p. either with a loading doseof 2 mg of 224G11 Mab/mouse and then twice a week with 1 mg ofantibody/mouse or with 3 injections of Temozolomide (D19, D26, D33) at 5mg/kg. A third group administered with the combine treatment was alsoincluded. Temozolomide was given by i.p. injections. Tumor volume wasmeasured twice a week and calculated by the formula:m/6×length×width×height and animal weights were monitored every day overthe period of Temozolomide treatment. Statistical analysis was performedat each measured time using either a t-test or a Mann-Whitney test. Bothsingle therapies and combined treatment displayed significant anti-tumoractivity compared to the control group (p≦002 from D22 to D32 (wherecontrol mice were euthanized for ethical reasons)). Results arerepresented in FIG. 48.

Combined treatment also demonstrate a significant anti-tumour growthactivity compared to single modality treatments (P≦002 from day 22 today 43 (where control mice were euthanized for ethical reasons) forTemozolomide and from day 29 to day 53 (last day of treatment) for224G11. Taken together, these data indicate that there is a benefit tocombine Temozolomide to an anti-c-Met treatment.

EXAMPLE 23 Spheroid Formation

As already described in Example 9 for other Mabs, we evaluate theability of 224G11 Mab to inhibit in vitro tumor growth in the U87-MGspheroid model. For that purpose, U87-MG cells grown as a monolayer weredetached with trypsine-EDTA and resuspended into complete cell culturemedia. Spheroids were initiated by inoculating 625 cells into singlewells of round bottom, 96 plates in DMEM-2.5% FCS. To prohibit celladhesion to a substratum, the plates were pre-coated with polyHEMA in95% ethanol and air dried at room temperature. The plates were incubatedunder standard cell culture conditions at 37° C., 5% CO₂ in humidifiedincubators. Purified monoclonal antibodies (10 μg/ml) were added after 4and 10 days of spheroid culture. Spheroids were kept in culture for 17days. Then, spheroid growth was monitored by measuring the area ofspheroids using automeasure module of axiovision software. Area wasexpressed in μm². 8-16 spheroids were evaluated for each condition.Spheroid size was measured before addition of antibodies, after 10 daysof culture and after 17 days of culture.

In those conditions, homogeneous spheroids were obtained and nostatistical difference was observed before addition of antibodies (FIG.49A).

As illustrated in FIGS. 49B-49D, isotype control, 9G4 did not affectedgrowth of spheroids after 10 or 17 days of culture. While addition of5D5 had no major effect on spheroid size, addition of either 224G11 and11E1 markedly inhibited tumor growth.

EXAMPLE 24 In Vitro Activity of Chimeric and Humanized Forms of 224G11in the Phospho-c-Met Assay

In order to compare the in vitro efficacy of murine, chimeric andhumanized forms in a functional assay, culture supernatants resultingfrom 224G11 hybridoma, and HEK293 transfected cells were dosed andtested as described in Example 3. Data summarized in FIG. 50 showed theexpected results for the unpurified morin antibody as already describedin FIG. 6B. Both chimeric and humanized unpurified antibodies displayeda comparable activity either when added alone (FIG. 50A) or whenincubated in presence of HGF (FIG. 50B).

EXAMPLE 25 Determination of Affinity Constants (KD) of Anti-c-MetAntibodies by Biacore Analysis

The binding affinity of purified 11E1 and 224G11 antibodies wasinvestigated by BIAcore X using recombinant c-Met-Extra-Cellular Domain(ECD) fused to an human IgG1 Fc domain (R&D Systems) as antigen (MW=129kDa). As both c-Met-Fc fusion proteins and antibodies are bivalentcompounds, Fab fragments of mAbs 11E1 and 224G11 (MW=50 kDa) weregenerated by papain cleavage, purified and used in this assay to avoidinterference with avidity parameter. For the assay, an anti-Taghistidine capture antibody was coated on CMS sensorships. The runningbuffer was HBS-EP, the flow rate was 30 μl/min and the test wasperformed at 25° C. Soluble c-Met (ECD_M1)2-Fc-(HHHHHH)2 antigen wascaptured on the sensorchip (around 270 RU), and the antibodies to betested were used in solution as analytes. The sensorship was regeneratedusing Glycine, HCl pH 1.5 buffer on both flowcells for half a minute.

FIG. 51 illustrates the principle of this analysis. The resultingkinetic parameters are summarized in the following table 4. Theyindicate that both 11E1 and 224G11 anti-c-Met antibodies bind therecombinant c-Met-Fc fusion protein with comparable affinities rangingabout 40 pM.

TABLE 4 K_(on1) × K_(on1) × 10⁻⁶ 10⁻⁶ Half-Life K [1/M · s] [1/M · s][h] [pM] 11E1 1.13 ± 0.01 4.68 ± 0.001 4.1 41.4 ± 0.5 Fab 224G11 2.04 ±0.01 7.79 ± 0.40  2.5 34.8 ± 1.9 Fab

EXAMPLE 26 In Vivo Activity of 224G11 on MDA-MB-231 Cells Co-ImplantedWith MRCS Cells as Human HGF Source on Athymic Nude Mice

MDA-MB-213 and MRCS cells from ATCC were both cultured in DMEM medium,10% FCS, 1% L-Glutamine. Cells were split two days before engraftmentbeing in exponential phase of growth. Five million MDA-MB-231 cells and500 000 MRCS cells were co-injected s.c. to Athymic nude mice. Twelvedays after implantation, tumors were measurable and animals were dividedinto groups of 6 mice with comparable tumor size. Mice were treated i.p.either with a loading dose of 2 mg of 224G11 Mab/mouse and then twice aweek with 1 mg of antibody/mouse. Tumor volume was measured twice a weekand calculated by the formula: m/6×length×width×height.

Results described in FIG. 52 showed a significant difference in mediantumors growth of mice treated with 224G 11 compared to the one of thecontrol group.

EXAMPLE 27 Complementary Elements on Humanization of Antibodies 227H1,11E1 and 224G11

General Procedure

Humanization of the anti-cMet antibodies were performed independentlyfor each chain and sequentially, regarding to the analysed amino acidsin each variable domain. The humanization process was evaluated in afirst attempt in an ELISA-based binding assay to recombinant Fc-cMet;binding activities the humanized antibodies being compared to therecombinant chimeric antibody. In a second attempt, anti-cMet humanizedantibodies were evaluated for their abilities to displace the Fc-cMetbinding onto plastic-coated recombinant HGF; this competition assayallowing the direct comparison of murine, chimeric and humanizedversions of the anti-cMet antibodies.

In FIGS. 53 and 54 are exemplified the typical anti-cMet bindingactivities of 227H1, 11E1 and 224G11 murine monoclonal antibodies.

FIG. 53 shows anti-cMet direct binding activities of detected purifiedmurine antibodies. In this assay, murine monoclonal anti-cMet antibodiesdisplay different but still dose-dependent anti-cMet binding activities.

FIG. 54 shows the HGF-cMet binding competition activities of purifiedmurine antibodies. The competition assay reveals reliable differencesbetween these anti-cMet monoclonal antibodies with a moderate, not fullbut reliable competitive activity for 11E1 monoclonal antibody whereasmurine 224G11 and 227H1 display similar pattern of competitiveactivities with a 100% of maximum of HGF binding displacement at highantibody concentration. The 224G11 monoclonal antibody showing the bestIC₅₀ value.

It is worthnoting that the direct binding activities of the murineantibodies do not reflect their intrinsic HGF-binding competitiveproperties.

These two assays were used to characterize the recombinant chimeric andhumanized versions of the murine anti-cMet antibodies. To this end,briefly, anti-cMet variable domains, either murine or humanized, werecloned into LONZA's pCONplus expression vectors series and recombinantIgG₁/κ-derived antibodies were expressed in CHO cells. Expressionculture supernatants were concentrated and extensively dialysed againstPBS and then dosed for expressed antibodies concentrations and directlyused to assess corresponding anti-cMet binding activities. Both directbinding and HGF-competition assays were assessed to better characterizerecombinant chimeric or humanized versions.

EXAMPLE 27-1 Humanization of 227H1 Heavy Chain Variable Domain

In order to identify the best human candidate for the CDR grafting, thehuman germline gene displaying the best identity with the 227H1 VHmurine sequence has been searched. With the help of the IMGT database,human IGHV1-2*02 V germline gene and human IGHJ4*01 J germline gene havethus been selected as receiving human sequences for the murine 227H1 VHCDRs.

FIG. 55 represents an amino acid alignment of the murine 227H1 VH domainwith the selected human framework. In the human FR lane, only the aminoacid that was found different from the 227H1 murine VH domain isdepicted. HZ3VH, HZ2VH and HZ1VH lanes correspond to implementedhumanized versions of the 227H1 VH domain with above (“changed in” lane)mentioned mutations clearly identified. The number under each proposedmutation corresponds to the rank at which said mutation will be done.

In a first series of experiments, we constructed and analysed theanti-cMet binding activities of the three first humanized versions ofthe 227H1 murine VH domain when expressed in combination with the 227H1chimeric light chain. Results obtained from the anti-cMet direct bindingassay are shown in FIG. 56. In this experiment, no differences in thebinding capabilities of the tested 227H1-derived chimeric or partiallyhumanized recombinant antibodies were observed. At this point, 26 out ofthe 32 amino acids that were found different between the murine 227H1 VHdomain and the selected human framework have been analysed and found notrelevant for anti-cMet binding activity of the 227H1 humanized VHdomain, when combined with the chimeric light chain.

In conjunction with a site-directed mutagenesis analysis of the last sixmurine residues in the HZ1VH humanized version of the 227H1 VH domain,we constructed an original HZ4VH <<full-IMGT humanized>> version andtested its anti-cMet binding properties. Results are given in FIG. 57for the direct binding assay and in FIG. 58 for the HGF bindingcompetition assay. It is worthnoting that both the recombinant chimericand humanized 227H1 versions display a better competitive activity thanthe parental murine antibody.

Nevertheless, given the experimental data obtained regarding theanti-cMet binding properties of the “full-IMGT” humanized 227H1 VHdomain, the resulting amino acid sequence depicted in FIG. 59 wasselected and a bioinformatic analysis was then performed to evaluate the<<humaness>> level of the so-called 227H1-HZ VH humanized variabledomain.

To this end a simple comparison of the frameworks sequences to humandatabase was performed using the IMGT tools. Given the humanizationlevel that we reached during this process, out of the 89 analysed aminoacids corresponding to the framework residues, 89 were found reliablewith a human origin. Only residues from the CDRs can be found different,but if so there are different from the corresponding human germlinegene, and are obviously at hypervariable positions. Based on the IMGTnumbering system and homology analysis tools, we first totally humanizedan antibody variable domain of murine origin.

EXAMPLE 27-2 11E1 Monoclonal Antibody Humanization

I—Humanization of 11E1 Heavy Chain Variable Domain

In order to identify the best human candidate for the CDR grafting, thehuman germline gene displaying the best identity with the 11E1 VH murinesequence has been searched. With the help of the IMGT database, humanIGHV1-46*01 V germline gene and human IGHJ4*03 J germline gene have thusbeen selected as receiving human sequences for the murine 11E1 VH CDRs.

FIG. 60 represents an amino acid alignment of the murine 11E1 VH domainwith the selected human framework. In the human FR lane, only the aminoacid that was found different from the 11E1 murine VH domain isdepicted. HZ VH3, HZ VH2 and HZ VH1 lanes correspond to implementedhumanized versions of the 11E1 VH domain with above (“changed in” lane)mentioned mutations clearly identified. The number under each proposedmutation corresponds to the rank at which said mutation will be done.

In a first series of experiments, we constructed and analysed theanti-cMet binding activities of the three first humanized versions ofthe 11E1 murine VH domain when expressed in combination with the 11E1chimeric light chain. Results obtained from the anti-cMet direct bindingassay are shown in FIG. 61. In this experiment, a similar bindingcapability of the tested 11E1-derived chimeric or partially humanizedrecombinant antibodies was observed. At this point, 19 out of the 24amino acids that were found different between the murine 11E1 VH domainand the selected human framework have been analysed and found notrelevant for anti-cMet binding activity of the 11E1 humanized VH domain,when combined with the chimeric light chain.

II—Humanization of 11E1 Light Chain Variable Domain

In order to identify the best human candidate for the CDR grafting, thehuman germline gene displaying the best identity with the 11E1 VL murinesequence has been searched. With the help of the IMGT database, humanIGKV3D-7*01 V germline gene and human IGKJ4*01 J germline gene have thusbeen selected as receiving human sequences for the murine 11E1 VL CDRs.

FIG. 62 represents an amino acid alignment of the murine 11E1 VL domainwith the selected human framework. In the human FR lane, only the aminoacid that was found different from the 11E1 murine VL domain isdepicted. HZ VL3, HZ VL2 and HZ VL1 lanes correspond to implementedhumanized versions of the 11E1 VL domain with above (“changed in” lane)mentioned mutations clearly identified. The number under each proposedmutation corresponds to the rank at which said mutation will be done.

In a first series of experiments, was constructed and the analysed theanti-cMet binding activities of the three first humanized versions ofthe 11E1 murine VL domain when expressed in combination with the 11E1chimeric heavy chain. Results obtained from the anti-cMet direct bindingassay are shown in FIG. 63. In this experiment, we observed similarbinding capabilities of the tested 11E1-derived chimeric or partiallyhumanized recombinant antibodies. At this point, 26 out of the 30 aminoacids that were found different between the murine 11E1 VL domain andthe selected human framework have been analysed and found not relevantfor anti-cMet binding activity of the 11E1 humanized VL domain, whencombined with the chimeric heavy chain.

III—Humanization of 11E1 Antibody

At this stage of the 11E1 monoclonal antibody humanization, thetheoretical resulting humanized antibody sequence contains only fiveoutside-CDRs residues coming from the parental murine VH domain and fouroutside-CDRs residues coming from the parental murine VL sequence (seeFIG. 60, lane HZ VH1 and FIG. 62, lane HZ VL1). It has then be decidedto immediately characterize the resulting combined heavy and light chainhumanized version of the 11E1 antibody. Results are given in FIG. 64 forthe anti-cMet direct binding assay.

In this experiment, it has been observed similar binding capabilitiesfor the tested 11E1-derived chimeric or humanized recombinantantibodies. Analysis of the HGF-binding competitive properties andsite-directed mutagenesis analysis of the contribution of the nine leftmurine residues remaining to be performed independently or incombination in this selected VH1/VL1 “pre-humanized” version of the 11E1monoclonal antibody.

EXAMPLE 27-3 224G11 Monoclonal Antibody Humanization

I—Humanization of 224G11 Heavy Chain Variable Domain

In order to identify the best human candidate for the CDR grafting, thehuman germline gene displaying the best identity with the 224G11 VHmurine sequence has been searched.

Regarding the high sequence homology between the 224G11 and the 227H1 VHdomains sequences, and as confirmed by the use of the IMGT databasetools, the same human IGHV1-2*02 V germline gene and human IGHJ4*01 Jgermline gene have thus been selected as receiving human sequences forthe murine 224G11 VH CDRs.

Based on this high homology, it has been decided to directly transferredthe humanization informations gained from the 227H1 VH domainhumanization (see Example 27) and we then designed a “full-IMGT”humanized version as depicted in FIG. 65 which represents an amino acidalignment of the murine 227H1 and 224G11 VH domains with the selectedhuman framework. In the human FR lane, only the amino acid that wasfound different from the 224G11 murine VH domain is depicted. HZVH0 lanecorresponds to <<full-IMGT>> humanized version of the 224G11 VH domainas obtained for the “full-IMGT” 227H1-HZVH domain.

The <<full-IMGT>> humanized version of the 224G11 murine VH domain hasthen been constructed and its anti-cMet binding activities wereanalysed, when expressed in combination with the 224G11 chimeric lightchain. Results obtained from the anti-cMet direct binding assay areshown in FIG. 66 while FIG. 67 illustrates the HGF binding competitionassay. Given the experimental data obtained regarding the anti-cMetbinding properties of the “full-IMGT” humanized 224G11 VH domain, theresulting amino acid sequence as depicted in FIG. 65 was selected and abioinformatic analysis was then performed to evaluate the <<humaness>>level of the so-called 224G 11-HZ VH0 domain.

Given the humanization strategy applied here, it has to be referred tothe Example 27 for the humaness analysis of the 224G11 HZ VH0 sequence.As described for the 227H1 VH domain humanization, we confirm thereliability of the IMGT numbering system and homology analysis tools,and also demonstrate the possibility of transferring the humanizationstrategy between antibodies under the limits of their intrinsichomology.

II—Humanization of 224G 11 Light Chain Variable Domain

In order to identify the best human candidate for the CDR grafting, thehuman germline gene displaying the best identity with the 224G11 VLmurine sequence has been searched. With the help of the IMGT databaseanalysis tools, two possible receiving human V regions for the murine224G11 VL CDRs were identified. Thus, two humanization strategies havebeen planed for the 224G11 VL domain. The first corresponds to aninitial trial for a human framework with a shorter CDR1 length(IGKV3-11*01), the second with a longer CDR1 length (IGKV4-1*01).

FIG. 68 represents an amino acid alignment of the murine 224G11 VLdomain with the two selected human frameworks. In the both shorter andlonger Hu-FR FR lanes, only the amino acid that was found different fromthe 224G11 murine VL domain is depicted. HZ VL3 and HZ VL6 lanescorrespond to basic humanized versions of the 224G11 VL domain withabove (“rank” lane) mentioned mutations clearly identified. The numberunder each proposed mutation corresponds to the rank at which saidmutation will be done whenever the basic “shorter” or “longer”CDR1-framework will be selected.

In a first set of experiments, the two basic humanized versions of the224G11 murine VL domain were constructed and their anti-cMet bindingactivities were analysed, when expressed in combination with the 224G11chimeric heavy chain. Results obtained from the anti-cMet direct bindingassay are shown in FIG. 69. In this experiment, a similar anti-cMetbinding activity was observed for the chimeric and HZ VL6(<<longer-CDR1>>) version whereas almost no binding was detected for theHZ VL3 (<<shorter-CDR1>>) recombinant 224G11-derived antibody.

In a second set of experiments, we constructed and analysed theanti-cMet binding activities of the implemented humanized versions ofthe HZ VL6-derived 224G11 VH domain when expressed in combination withthe 224G11 chimeric heavy chain. Two additional humanized form wasanalysed; in the HZ VL5 version the seven residues from the third group(rank 3) are humanized and in the HZ VL4 version the four left residuesfrom the first group (rank 1 residues) only remained murine. Resultsobtained from the anti-cMet direct binding assay are shown in FIG. 70.In this experiment, no differences in the binding capabilities of thetested 224G11-derived chimeric or partially humanized recombinantantibodies were observed. At this point, 18 out of the 22 amino acidsthat were found different between the murine 224G11 VL domain and theselected <<longer-CDR1>> human framework have been analysed and foundnot relevant for anti-cMet binding activity of the 224G11 humanized VLdomain, when combined with the chimeric heavy chain.

It has then be tested the HZ VL4 humanized version of the 224G11 VLdomain in the HGF binding competition assay. As shown in FIG. 71, theresults obtained demonstrate the similar competitive activity of murineand recombinant chimeric and HZ VL4 humanized 224G11-derived antibodies.

At this stage of the 224G11 VL domain humanization, the resultingsequence contains only four outside-CDRs residues coming from the murineparental sequence. As shown in FIG. 72, these four §-labelled residuesare L4, M39, H40 and R84.

Based on the IMGT numbering system and homology analysis tools, wedemonstrated that human framework displaying structural differences interm of CDR length may still be suitable in a humanization process. Ithas then been decided to characterize the resulting heavy and lightchain humanized version of the 224G11 antibody. Site-directedmutagenesis analysis of the contribution of the remaining four murineresidues being then performed when expressed in combination with the VH0humanized version of the heavy chain.

III—Humanization of 224G11 Antibody

In a first series of experiments, we constructed and analysed theanti-cMet binding activities of the fully humanized version of the224G11 antibody. This recombinant version encompass both VH0 and VL4humanized VH and VL domains respectively. Results obtained from theanti-cMet direct binding assay are shown in FIG. 73. In this experiment,the fully human 224G11 anti-cMet binding activity was found similar tothat of <<single-chain>> humanized and chimeric recombinant 224G11versions.

It has then been tested the fully humanized version of the 224G11 VLdomain in the HGF binding competition assay. The results obtained asshown in FIG. 74 demonstrate the similar competitive activity ofparental murine and recombinant chimeric and fully humanized224G11-derived antibodies.

At this stage of the 224G11 antibody humanization, the resultingsequence contains only four outside-CDRs residues coming from the murineparental light chain variable domain sequence. We then analysedsite-directed mutagenesis single variants of the VL4 humanized VL domainwhen expressed in combination with the VH0 humanized version of theheavy chain. As exemplified in FIG. 75 for the direct binding assay weidentified potential relevant residues among the four tested, being M39and H40.

It has been decided to analyse multiple mutants of the HZ VL4 humanized224G11 VL domain when expressed in combination with the HZ VH0 humanized224G11 VH domain. As shown in FIG. 76 for the direct binding assay andin FIG. 77 for the HGF binding competition assay, multiple amino acidsmutants of the VL4 domain were analysed to identify the best humanizedcombination. Based on the single mutants analysis, it has been focusedon double and triple mutants that may exhibits the best anti-cMetactivities. The VH0/VL4-2x mutant correspond to the HZ VH0 224G11humanized VH domain expressed with the HZ VL4 224G11 humanized VL domainwith the double mutation L4M/R84G. The VH0/VL4-3x mutant correspond tothe HZ VH0 224G11 humanized VH domain expressed with the HZ VL4 224G11humanized VL domain with the triple mutation L4M/M39L/R84G.

Given the experimental data obtained regarding the anti-cMet bindingproperties of the fully humanized 224G11 antibody the bioinformaticanalysis of both heavy and light chain variable domains sequences wasthen performed to evaluate the <<humaness>> level of the VH0/VL4-2x andVH0/VL4-3x best humanized versions. It has been previously demonstratedthe “full-IMGT” humanization of the VH0 224G11 VH domain. Regarding thehumaness level of the VL4-2x and -3x 224G11 humanized VL domainversions, they only contain murine residues M39 and/or H40. These twopotential key residues are located at the end of the CDR1, M39 being theN-terminal CDR anchor. Given the CDR length problem that we faced duringthe 224G11 VL domain humanization, and considering those positions aspart of the Kabat definition of the VL CDR1, the humaness level of thefully humanized 224G11 antibody should display a strongly reducedimmunogenicity due to the minimal conserved murine residues.

1.-128. (canceled)
 129. An isolated anti-cMet antibody comprising: aheavy chain comprising complementarity-determining region (CDR) CDR-H1,CDR-H2, and CDR-H3 comprising, respectively, SEQ ID Nos. 7, 8, and 9;and a light chain comprising CDR-L1, CDR-L2, and CDR-L3 comprising,respectively, SEQ ID Nos. 15, 16, and
 17. 130. The isolated anti-cMetantibody according to claim 129, comprising a heavy chain variableregion comprising SEQ ID No. 20 and a light chain variable regioncomprising SEQ ID No.
 23. 131. The isolated anti-cMet antibody accordingto claim 129, wherein the antibody is a monoclonal antibody.
 132. Theisolated anti-cMet antibody according to claim 131, wherein the antibodyis a chimeric antibody comprising light chain and heavy chain constantregions derived from an antibody of a species heterologous to mouse.133. The isolated anti-cMet chimeric antibody according to claim 132,wherein the species heterologous to mouse is human.
 134. The isolatedanti-cMet antibody according to claim 133, wherein the light chainconstant region derived from the antibody of human species is a kapparegion and the heavy chain constant region derived from the antibody ofhuman species is chosen from the gamma-1, gamma-2, and gamma-4 regions.135. A pharmaceutical composition comprising a pharmaceuticallyacceptable excipient and an antibody according to claim
 129. 136. Thepharmaceutical composition according to claim 135, wherein the antibodyis a 223C4 antibody produced by murine hybridoma number 1-3786 depositedat the CNCM, Institut Pasteur, Paris, on on Jul. 6,
 2007. 137. Thepharmaceutical composition according to claim 135, wherein the antibodycomprises a heavy chain variable region comprising SEQ ID No. 20 and alight chain variable region comprising SEQ ID No.
 23. 138. Thepharmaceutical composition according to claim 137, wherein the antibody:inhibits both ligand-dependent and ligand-independent activation ofc-Met; and/or inhibits at least 50% of tumoral cell proliferation for atleast one tumor type; and/or inhibits c-Met dimerization.
 139. Theisolated anti-cMet antibody of claim 129, wherein the antibody inhibitsboth ligand-dependent and ligand-independent activation of c-Met. 140.The isolated anti-cMet antibody of claim 139, wherein the antibodyinhibits c-Met dimerization.
 141. The isolated anti-cMet antibody ofclaim 140, wherein the antibody inhibits at least 50% of tumoral cellproliferation for at least one tumor type.
 142. The isolated anti-cMetantibody of claim 129, wherein the antibody inhibits c-Met dimerization.143. The isolated anti-cMet antibody according to any claim
 138. 144. Anantibody secreted by the hybridoma deposited at the CNCM, InstitutPasteur, Paris, on Jul. 6, 2007, under the number I-3786.
 145. Theisolated anti-cMet antibody of claim 130, wherein the antibody inhibitsboth ligand-dependent and ligand-independent activation of cMet. 146.The isolated anti-cMet antibody of claim 145, wherein the antibodyinhibits cMet dimerization.
 147. The isolated anti-cMet antibody ofclaim 147, wherein the antibody inhibits at least 50% of tumoral cellproliferation for at least one tumor type.
 148. The isolated anti-cMetantibody of claim 130, wherein the antibody inhibits c-Met dimerization.149. The isolated anti-cMet antibody according to claim
 145. 150. Theisolated anti-cMet antibody of claim 129, wherein the antibody: inhibitsboth ligand-dependent and ligand-independent activation of c-Met; and/orinhibits at least 50% of tumoral cell proliferation for at least onetumor type; and/or inhibits c-Met dimerization.
 151. The isolatedanti-cMet antibody of claim 130, wherein the antibody: inhibits bothligand-dependent and ligand-independent activation of c-Met; and/orinhibits at least 50% of tumoral cell proliferation for at least onetumor type; and/or inhibits c-Met dimerization.
 152. A cMet-bindingfragment of the isolated anti-cMet antibody according to claim
 129. 153.The cMet-binding fragment of claim 152, wherein the fragment is adivalent fragment.
 154. The cMet-binding fragment of claim 152, whereinthe fragment is a F(ab′)₂ fragment.
 155. The cMet-binding fragment ofclaim 152, wherein the fragment is a scFv fragment.
 156. A cMet-bindingfragment of the isolated anti-cMet antibody according to claim
 143. 157.The cMet-binding fragment of claim 156, wherein the fragment is adivalent fragment.
 158. The cMet-binding fragment of claim 156, whereinthe fragment is a F(ab′)₂ fragment.
 159. The cMet-binding fragment ofclaim 156, wherein the fragment is a scFv fragment.
 160. A cMet-bindingfragment of the isolated anti-cMet antibody according to claim
 149. 161.The cMet-binding fragment of claim 160, wherein the fragment is adivalent fragment.
 162. The cMet-binding fragment of claim 160, whereinthe fragment is a F(ab′)₂ fragment.
 163. The cMet-binding fragment ofclaim 160, wherein the fragment is a scFv fragment.
 164. The isolatedanti-cMet antibody of claim 129, wherein said antibody is coupledchemically to a cytotoxic agent.
 165. The isolated anti-cMet antibody ofclaim 164, wherein said cytotoxic agent is chosen from alkylatingagents, anti-metabolites, anti-tumor antibiotics, mitotic inhibitors,chromatin function inhibitors, anti-angiogenesis agents, anti-estrogenicagents, anti-androgen agents, and immunomodulators.
 166. The isolatedanti-cMet antibody of claim 164, wherein said cytotoxic agent is amitotic inhibitor.
 167. The pharmaceutical composition according toclaim 135, wherein said antibody is coupled chemically to a cytotoxicagent.
 168. The pharmaceutical composition according to claim 167,wherein said cytotoxic agent is chosen from alkylating agents,anti-metabolites, anti-tumor antibiotics, mitotic inhibitors, chromatinfunction inhibitors, anti-angiogenesis agents, anti-estrogenic agents,anti-androgen agents, and immunomodulators.
 169. The pharmaceuticalcomposition according to claim 31, wherein said cytotoxic agent is amitotic inhibitor.
 170. The cMet-binding fragment according to claim152, wherein said cMet-binding fragment is coupled chemically to acytotoxic agent.
 171. The cMet-binding fragment according to claim 170,wherein said cytotoxic agent is chosen from alkylating agents,anti-metabolites, anti-tumor antibiotics, mitotic inhibitors, chromatinfunction inhibitors, anti-angiogenesis agents, anti-estrogenic agents,anti-androgen agents, and immunomodulators.
 172. The cMet-bindingfragment according to claim 170, wherein said cytotoxic agent is amitotic inhibitor.
 173. The cMet-binding fragment according to claim156, wherein said cMet-binding fragment is coupled chemically to acytotoxic agent.
 174. The cMet-binding fragment according to claim 173,wherein said cytotoxic agent is chosen from alkylating agents,anti-metabolites, anti-tumor antibiotics, mitotic inhibitors, chromatinfunction inhibitors, anti-angiogenesis agents, anti-estrogenic agents,anti-androgen agents, and immunomodulators.
 175. The cMet-bindingfragment according to claim 173, wherein said cytotoxic agent is amitotic inhibitor.
 176. The cMet-binding fragment according to claim160, wherein said cMet-binding fragment is coupled chemically to acytotoxic agent.
 177. The cMet-binding fragment according to claim 176,wherein said cytotoxic agent is chosen from alkylating agents,anti-metabolites, anti-tumor antibiotics, mitotic inhibitors, chromatinfunction inhibitors, anti-angiogenesis agents, anti-estrogenic agents,anti-androgen agents, and immunomodulators.
 178. The cMet-bindingfragment according to claim 176, wherein said cytotoxic agent is amitotic inhibitor.
 179. An isolated anti-cMet antibody, or cMet-bindingfragment thereof, wherein the antibody or fragment thereof binds anepitope on cMet that is recognized by the 223C4 antibody produced bymurine hybridoma 1-3786 deposited at the CNCM, Institut Pasteur, Paris,on Jul. 6, 2007, and inhibits both ligand-dependent andligand-independent activation of c-Met.
 180. The isolated anti-cMetantibody of claim 179, wherein the antibody inhibits c-Met dimerization.181. The isolated anti-cMet antibody of claim 180, wherein the antibodyinhibits at least 50% of tumoral cell proliferation for at least onetumor type.
 182. The isolated anti-cMet antibody of claim 129, whereinthe antibody: inhibits both ligand-dependent and ligand-independentactivation of c-Met; and/or inhibits at least 50% of tumoral cellproliferation for at least one tumor type; and/or inhibits c-Metdimerization.
 183. The isolated anti-cMet antibody according to claim179, wherein the antibody does not bind to the semaphorin (SEMA) domainof cMet.